Ligand-drug conjugates, conjugation linkers, and uses thereof

Abstract

It provides the general formula Pc-(L-D). n The present invention represents a class of ligand-drug conjugates or pharmaceutically acceptable salts thereof, and also provides conjugation linkers as shown in formula (L), which can be chemically conjugated to thiol groups on cysteine ​​residues with high efficiency and selectivity, resulting in antibody-drug conjugates with good stability.

Description

Ligand-drug conjugates, conjugated linkers and uses thereof

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefits and priority of patent application No. 202311515509.5 filed with the State Intellectual Property Office of China on November 14, 2023 and patent application No. 202410173706.1 filed with the State Intellectual Property Office of China on February 6, 2024, the entire contents of which are hereby incorporated by reference into this document in their entirety. Technical Field

[0003] The present disclosure belongs to the field of biomedicine and relates to a class of ligand-drug conjugates with novel structures and their conjugated linkers, pharmaceutical compositions containing the conjugates, and uses of the conjugates as anti-tumor drugs. Background Art

[0004] Antibody-drug conjugates (ADCs) are a new form of drug therapy in recent years. Structurally, ADCs consist of a targeting antibody, a pharmacologically active payload, and a linker chemically connecting the antibody and payload. Mechanistically, ADCs leverage the antibody's specific antigen recognition to enhance ADC accumulation in lesion tissues where the antigen is highly expressed. Through endocytosis and lysosomal degradation, the active payload is released into the lesion, ultimately producing its pharmacological effect. Simultaneously, ADCs maintain a relatively stable state within the systemic circulation, allowing only a small amount of the payload to be released into the systemic circulation compared to the lesion tissue, thereby minimizing the systemic toxicity of the highly active payload. This selective release mechanism further enhances therapeutic efficacy and reduces the risk of toxicity. The linker, acting as a bridge between the antibody and the payload, plays a crucial role in controlling the payload's release into target tissues and maintaining its stability in the systemic circulation.

[0005] In the ADC linker, the part that connects the small molecule to the antibody through a chemical bond is called a coupling linker. The coupling methods with antibodies can be roughly divided into coupling with natural amino acid residues on the antibody (such as lysine, cysteine, etc.) and site-specific coupling through engineered antibody modification (such as N297 glycosyl coupling, non-natural amino acid coupling, etc.). Different antibody coupling methods have different corresponding coupling linkers. Among the ADCs currently on the market, coupling through the sulfhydryl group on cysteine ​​is one of the most commonly used coupling methods. Typically, antibodies contain four pairs of interchain disulfide bonds, and eight sulfhydryl-containing cysteines can be obtained through reduction. With the help of the nucleophilic activity of the sulfhydryl group, a chemical reaction can be carried out with the coupling linker to connect the antibody and the linker-payload together. Among them, the most commonly used coupling linker is maleimide, which can efficiently form a carbon-sulfur bond with the sulfhydryl group on cysteine ​​through a Michael addition reaction, thereby connecting the payload to the antibody. For example, Adcetris, Enhertu, etc. are ADC products obtained by maleimide-cysteine ​​coupling.

[0006] Summary of the Invention

[0007] In a first aspect, the present disclosure provides a ligand-drug conjugate or a pharmaceutically acceptable salt thereof, the general structural formula of which is Pc-(LD) n ,

[0008] in,

[0009] Pc is the ligand unit;

[0010] D is the drug unit;

[0011] L is a linker unit of the following structure, with the a-terminal end connected to the ligand unit,

[0012] in,

[0013] X 1 、X 2 、X 3 Each independently selected from N or CR a1 , and X 1 、X 2 、X 3 At least one of N, the R a1 is selected from H, halogen, CN, C1-C3 alkyl, C2-C3 alkenyl, C2-C3 alkynyl, C3-C6 cycloalkyl or 4-7 membered heterocyclyl;

[0014] n1 and n2 are each independently selected from 0, 1, and 2;

[0015] X 4 、X5 Each independently selected from C(=O), C(R b1 )(R b2 ), O, S, S(=O), S(=O)2, NR b3 or any combination thereof, the R b1 、R b2 、R b3 Each independently selected from H or R c1 , or R b1 、R b2 Together with the carbon atoms to which they are attached, they form a C3-C6 cycloalkyl or a 4-7 membered heterocyclic group, wherein the C3-C6 cycloalkyl or the 4-7 membered heterocyclic group is optionally substituted by one or more R c1 replace;

[0016] Every R c1 Independently selected from halogen, CN, C1-C6 alkyl, C3-C6 cycloalkyl or hydrophilic group;

[0017] L a Selected from R b4 、R b5 Each is independently selected from H, halogen, CN, C1-C3 alkyl, OH, O(C1-C3 alkyl), NH2, NH(C1-C3 alkyl) or N(C1-C3 alkyl)2, or R b4 、R b5 Together with the carbon atoms to which they are connected, they form a C3-C6 cycloalkyl group or a 4-7 membered heterocyclic group, m4 is an integer selected from 1 to 10, L a The c-terminus and L b connect;

[0018] L b Select from chemical bonds or m5 is selected from integers of 0 to 24, m6 is selected from integers of 1 to 10, and each R b6 、R b7 、R b8 Each independently selected from H or R c6 , the R c6 is selected from halogen, CN, C1-C6 alkyl, C3-C6 cycloalkyl or hydrophilic group, L b The d end and L c connect;

[0019] L c Selected from Y 1 -Y 2 -Y 3 , Y 1 Select from chemical bonds or m7 is an integer selected from 1 to 6, Y 2is selected from a peptide residue consisting of 2-10 amino acids, Y 3 Selected from L c The b-end is connected to the drug unit D;

[0020] n is a real number from 1 to 16.

[0021] In some embodiments, X 1 、X 2 All selected from N, X 3 Selected from CH.

[0022] In some embodiments, n1 and n2 are each independently selected from 0 or 1.

[0023] In some embodiments, n1 is selected from 1 and n2 is selected from 0.

[0024] In some embodiments, X 4 、X 5 Each independently selected from C(=O) or C(R b1 )(R b2 ), the R b1 、R b2 Each independently selected from H or R c1 , or R b1 、R b2 Together with the carbon atoms to which they are attached, they form a C3-C6 cycloalkyl or a 4-7 membered heterocyclic group, wherein the C3-C6 cycloalkyl or the 4-7 membered heterocyclic group is optionally substituted by one or more R c1 replace.

[0025] In some embodiments, X 4 Selected from C(=O), X 5 Selected from C(R b1 )(R b2 ), the R b1 、R b2 Each independently selected from H or R c1 , or R b1 、R b2 Together with the carbon atoms to which they are attached, they form a C3-C6 cycloalkyl or a 4-7 membered heterocyclic group, wherein the C3-C6 cycloalkyl or the 4-7 membered heterocyclic group is optionally substituted by one or more R c1 replace.

[0026] In some embodiments, X 4 Selected from C(=O), X 5 Selected from C(R b1 )(R b2 ), the R b1 、R b2 Each independently selected from H or R c1 , or Rb1 、R b2 Together with the carbon atoms to which they are attached, they form a C4-C6 cycloalkyl group or a 4-6 membered heterocyclic group containing 1 O atom or 1 N atom, wherein the 4-6 membered heterocyclic group containing 1 O atom or 1 N atom is optionally replaced by one R c1 Replace; each of R c1 Independently selected from

[0027] In some embodiments, L b Selected from chemical bonds, wherein m5 is selected from an integer of 1 to 16, for example, m5 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16, m6 is selected from an integer of 1 to 6, for example, m6 is selected from 1, 2, 3, 4, 5 or 6, R b7 、R b8 Each independently selected from H or R c6 , L b The d end and L c connect.

[0028] In some embodiments, L b Select from chemical bonds or where R b7 is H, and R b8 H or R c6 , L b The d end and L c connect.

[0029] In some embodiments, each R c1 and R c6 Each is independently selected from halogen, CN, C1-C6 alkyl, C3-C6 cycloalkyl or a hydrophilic group, the hydrophilic group comprising one or more of the following atomic groups or structural fragments: C(=O), OH, O(C1-C3 alkyl), NH2, NH(C1-C3 alkyl), N(C1-C3 alkyl)2, C1-C4 alkyl-O, NHC(=O), N(CH3)C(=O), C(=O)NH2, S(=O)2NH2, C(=O)NH(C1-C3 alkyl), S(=O)2NH(C1-C3 alkyl), C(=O)N(C1-C3 alkyl)2, S(=O)2N(C1-C3 alkyl)2,

[0030] In some embodiments, each R c1 and R c6 independently selected from halogen, CN, C1-C6 alkyl, C3-C6 cycloalkyl or W1 -W 2 -W 3 -W 4 -W 5 , each W 1 、W 2 、W 3 、W 4 Each independently selected from a chemical bond, A-(C1-C4 alkyl-O) m1 or [A-(CR d1 R d2 ) m2 -NR d3 ] m3 -B, each A and B is independently selected from a chemical bond, C1-C 10 Alkylene, C3-C6 cycloalkylene, 4-7 membered heterocyclylene, O, S, NR d4 、C=O、C(=O)NR d5 、S(=O)2、S(=O)2NR d6 、S(=O)(=NR d7 ), or any combination thereof, the R d1 、R d2 、R d3 、R d4 、R d5 、R d6 、R d7 、R d8 Each is independently selected from H, C1-C6 alkyl, C3-C6 cycloalkyl, 4-7 membered heterocyclyl or (C1-C4 alkyl-O) k1 -C1-C6 alkyl, the C1-C6 alkyl, C3-C6 cycloalkyl or 4-7 membered heterocyclyl is optionally substituted by halogen, CN, OH, O(C1-C3 alkyl), NH2, NH(C1-C3 alkyl), N(C1-C3 alkyl)2, C(=O)NH2, NHC(=O)NH2 or COOH, m1 is selected from an integer of 0 to 30, m2 is selected from an integer of 1 to 5, m3 and k1 are each independently selected from an integer of 1 to 30, W 5 Selected from H, C1-C6 alkyl, C3-C6 cycloalkyl, 4-7 membered heterocyclyl, OH, O(C1-C3 alkyl), NH2, NH(C1-C3 alkyl), N(C1-C3 alkyl)2, C(=O)NH2, S(=O)2NH2, C(=O)NH(C1-C3 alkyl), S(=O)2NH(C1-C3 alkyl), C(=O)N(C1-C3 alkyl)2, S(=O)2N(C1-C3 alkyl), In some embodiments, each R c1 and R c6 Independently selected from W1 -W 2 -W 3 -W 4 -W 5 , W 1 、W 2 are all selected from chemical bonds, W 3 Selected from chemical bonds or A-(C1-C4 alkyl-O) m1 , W 4 Selected from A-(C1-C4 alkyl-O) m1 or [A-(CR d1 R d2 ) m2 -NR d3 ] m3 -B, each A and B is independently selected from a chemical bond, C1-C 10 Alkylene, C3-C6 cycloalkylene, 4-7 membered heterocyclylene, O, S, NR d4 、C=O、C(=O)NR d5 、S(=O)2、S(=O)2NR d6 、S(=O)(=NR d7 ), or any combination thereof, the R d1 、R d2 、R d3 、R d4 、R d5 、R d6 、R d7 、R d8 Each is independently selected from H, C1-C6 alkyl, C3-C6 cycloalkyl, 4-7 membered heterocyclyl or (C1-C4 alkyl-O) k1 -C1-C6 alkyl, the C1-C6 alkyl, C3-C6 cycloalkyl or 4-7 membered heterocyclyl is optionally substituted by halogen, CN, OH, O(C1-C3 alkyl), NH2, NH(C1-C3 alkyl), N(C1-C3 alkyl)2, C(=O)NH2, NHC(=O)NH2 or COOH, m1 is selected from an integer of 0 to 30, m2 is selected from an integer of 1 to 5, m3 and k1 are each independently selected from an integer of 1 to 30, W 5 Selected from H, C1-C6 alkyl, C3-C6 cycloalkyl, 4-7 membered heterocyclyl, OH, O(C1-C3 alkyl), NH2, NH(C1-C3 alkyl), N(C1-C3 alkyl)2, C(=O)NH2, S(=O)2NH2, C(=O)NH(C1-C3 alkyl), S(=O)2NH(C1-C3 alkyl), C(=O)N(C1-C3 alkyl)2, S(=O)2N(C1-C3 alkyl)2, In some embodiments, each Rc1 Independently selected from A-(C1-C4 alkyl-O) m1 -W 5 or [C(=O)-CH2-NR d3 ] m3 -BW 5 , A is selected from a chemical bond or C=O, R d3 is selected from C1-C6 alkyl, B is selected from C1-C 10 Alkylene, C=O or a combination thereof C(=O)-C1-C 10 Alkylene, W 5 is selected from C1-C6 alkyl, C(=O)NH2 or S(=O)2NH2, and m1 and m3 are independently selected from integers of 1 to 24.

[0031] In some embodiments, each R c1 Independently selected from

[0032] In some embodiments, each R c1 Independently selected from

[0033] In some embodiments, each R c6 Independently selected from W 1 -W 2 -W 3 -W 4 -W 5 , W 1 、W 2 are all selected from chemical bonds, W 3 Selected from chemical bonds or A-(C1-C4 alkyl-O) m1 , W 4 Selected from A-(C1-C4 alkyl-O) m1 or [C(=O)-CH2-NR d3 ] m3 -B, each A and B is independently selected from a chemical bond, a C1-C6 alkylene, NR d4 ,C(=O),C(=O)NH,S(=O)2,S(=O)2NR d6 、S(=O)(=NR d7 ), or any combination thereof, the R d1 、R d2 、R d3 、R d4 、R d6 、R d7 、R d8Each independently selected from H, C1-C6 alkyl or (C1-C4 alkyl-O) k1 -C1-C6 alkyl, m1 is selected from an integer of 0 to 24, m2 is selected from an integer of 1 to 5, m3 and k1 are each independently selected from an integer of 1 to 24, W 5 Selected from C1-C6 alkyl, NH2,

[0034] In some embodiments, R c6 Selected from

[0035] In some embodiments, R c6 Selected from

[0036] In some embodiments, L a Selected from R b4 、R b5 Each is independently selected from H, m4 is selected from an integer of 1 to 6, for example, m4 is selected from 1, 2, 3, 4, 5 or 6, L a The c-terminus and L b In some embodiments, L a Selected from R b4 、R b5 Each is independently selected from H, m4 is selected from an integer of 1 to 6, for example, m4 is selected from 1, 2, 3, 4, 5 or 6, L a The c-terminus and L b connect.

[0037] In some embodiments, L c Selected from Y 1 -Y 2 -Y 3 , Y 1 Select from chemical bonds or Y 2 A dipeptide, tripeptide or tetrapeptide residue selected from Val-Cit, Val-Ala, Gly-Gly-Phe-Gly, Ala-Ala-Ala, Y 3 Selected from L c The b-end of the drug unit is connected to the drug unit D.

[0038] In some embodiments, L c Selected from Y 1 -Y 2 -Y 3 , Y 1 Select from chemical bonds or Y 2 A dipeptide or tetrapeptide residue selected from Val-Cit, Val-Ala, Gly-Gly-Phe-Gly, Y 3 Selected from L c The b-end of the drug unit is connected to the drug unit D.

[0039] In some embodiments, L c Selected from

[0040] In some embodiments, L c Selected from

[0041] In some embodiments, the linker unit L is selected from the following structures:

[0042] In some embodiments, the Drug unit D is selected from a cytotoxic drug and a protein degrader.

[0043] In some embodiments, the cytotoxic drug is selected from a microtubule inhibitor, a DNA damaging agent or a topoisomerase inhibitor, the microtubule inhibitor includes but is not limited to dolastatin, auristatin, maytansine, tubulysins and cryptomycins, the DNA damaging agent includes but is not limited to PBD, duocarmycin and calicheamicin drugs, the topoisomerase inhibitor includes but is not limited to camptothecin drugs.

[0044] In some embodiments, the cytotoxic drugs include but are not limited to camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotecan, rubitecan, dactinomycin, doxorubicin, duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin, etoposide, vincristine, vinblastine, paclitaxel, docetaxel, cabazitaxel, maytansine, maytansine-like, DM1, DM3, DM4, calicheamicin, MMAE, MMAF, and Tubulysin D.

[0045] In some embodiments, the Drug unit D is selected from a topoisomerase I inhibitor or an auristatin.

[0046] In some embodiments, the protein degrader is selected from a GSPT1 protein degrader.

[0047] In some embodiments, D is selected from

[0048] In some embodiments, the disclosed formula is Pc-(LD) n The ligand-drug conjugate or a pharmaceutically acceptable salt thereof is selected from the following compounds or a pharmaceutically acceptable salt thereof:

[0049] In some embodiments, the Ligand unit Pc can be selected from a polypeptide, an antibody, or an antigen-binding fragment thereof.

[0050] In some embodiments, the ligand unit Pc can specifically bind to one or more antigens selected from the following group: HER2 (ErbB2), p95HER2, HER3 (ErbB3), CD3, CD16, ROR1, DLL3, CDH6, CD70, CD5, CD20, BCMA, EGFR, VEGF, Trop-2, Claudin6, Claudin18.2, c-MET, CD30, MUC1, Nectin-4, CD22, CD74, CD19, CD79b, MSLN, TOPO2, EpCAM, CEACAM-5, Mesothelin, PD-L1, PSMA, ROR1, ROR2, CD25, CD33, CD123, FLT3, CD174, CD166, CD326, CD71, LIV-1, MUC16, ENPP3, TDGF1, ETBR, TIM1, TIM3, LRRC15, CanAg / AFP, FAP, SLITRK6, KIT / CD117, STEAP1, SLAMF7 / CS1, NaPi2B / SLC34A2, GPNMB, AXL, B7-H3(CD276), PTK7 / CCK4, PRLR , EFNA4, 5T4, NOTCH3, CD142, CA6, GPR20, EphA2, LYPD3, FGFR2, FGFR3, FRα, CEACAMs, GCC, IntegrinAv, CAIX, P-cadherin, GD3, cadherin6, LAMP1, CD56, CD37, CD47, CD138 or CD352.

[0051] In some embodiments, the Ligand unit Pc can specifically bind to HER2.

[0052] In some embodiments, the Pc comprises an antibody or antigen-binding fragment thereof that specifically binds to HER2; the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein:

[0053] (1) the heavy chain variable region comprises HCDR1 of SEQ ID NO.5, HCDR2 of SEQ ID NO.6, and HCDR3 of SEQ ID NO.7, or / and the light chain variable region comprises LCDR1 of SEQ ID NO.8, LCDR2 of SEQ ID NO.9, and LCDR3 of SEQ ID NO.10; or,

[0054] (2) the heavy chain variable region comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to (1) in each CDR, or has at most 3, 2, or 1 insertion, deletion or substitution mutations, and / or the light chain variable region comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to (1) in each CDR, or has at most 3, 2, or 1 insertion, deletion or substitution mutations.

[0055] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein:

[0056] (1) the heavy chain variable region comprises the sequence shown in SEQ ID NO. 1, or / and the light chain variable region comprises the sequence shown in SEQ ID NO. 2; or,

[0057] (2) The heavy chain variable region and / or the light chain variable region have a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the heavy chain variable region and / or the light chain variable region in any of the groups in group (1) above, or a sequence in which at most 3, 2 or 1 insertion, deletion or substitution mutations occur.

[0058] In some embodiments, the Ligand unit Pc can specifically bind to CDH6.

[0059] In some embodiments, the Pc comprises an antibody or antigen-binding fragment thereof that specifically binds to CDH6; the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein:

[0060] (1) the heavy chain variable region comprises HCDR1 of SEQ ID NO. 17, HCDR2 of SEQ ID NO. 18, and HCDR3 of SEQ ID NO. 19, or / and the light chain variable region comprises LCDR1 of SEQ ID NO. 20, LCDR2 of SEQ ID NO. 21, and LCDR3 of SEQ ID NO. 22; or,

[0061] (2) the heavy chain variable region comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to (1) in each CDR, or has at most 3, 2, or 1 insertion, deletion or substitution mutations, and / or the light chain variable region comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to (1) in each CDR, or has at most 3, 2, or 1 insertion, deletion or substitution mutations.

[0062] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein:

[0063] (1) the heavy chain variable region comprises the sequence shown in SEQ ID NO. 15, or / and the light chain variable region comprises the sequence shown in SEQ ID NO. 16; or,

[0064] (2) The heavy chain variable region and / or the light chain variable region have a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the heavy chain variable region and / or the light chain variable region in any of the groups in group (1) above, or a sequence in which at most 3, 2 or 1 insertion, deletion or substitution mutations occur.

[0065] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain constant region sequence and / or a light chain constant region sequence; optionally, the heavy chain constant region and / or light chain constant region comprises a complete constant region sequence or a fragment thereof, and the constant region fragment comprises CH1, hinge region, CH2, CH3 or Fc; optionally, the heavy chain constant region is selected from human or mouse IgG1, IgG2, IgG3 or IgG4 constant region, or / and, the light chain constant region is selected from human or mouse kappa constant region or lamda constant region; optionally, the antibody or antigen-binding fragment comprises a complete heavy chain and a light chain, the heavy chain consists of the VH and the heavy chain constant region, the heavy chain constant region comprises the sequence shown in SEQ ID NO.3, and the light chain consists of the VL and the light chain constant region, and the light chain constant region comprises the sequence shown in SEQ ID NO.4.

[0066] In some embodiments, the antibody or antigen-binding fragment is:

[0067] (1) Chimeric antibodies or fragments thereof;

[0068] (2) humanized antibodies or fragments thereof; and / or,

[0069] (3) fully human antibodies or fragments thereof;

[0070] Preferably, the antibody or antigen-binding fragment is selected from a monoclonal antibody, a polyclonal antibody, a natural antibody, an engineered antibody, a monospecific antibody, a multispecific antibody (e.g., a bispecific antibody), a monovalent antibody, a multivalent antibody, a full-length antibody, an antibody fragment, a naked antibody, a conjugated antibody, a humanized antibody, a fully human antibody, Fab, Fab', F(ab')2, Fd, Fv, scFv, a diabody or a single-domain antibody (sdAb).

[0071] In some embodiments, the antigen-binding fragment is selected from one or more of F(ab)2, Fab', Fab, Fv, scFv, bispecific antibody, nanobody and antibody minimal recognition unit.

[0072] In some embodiments, the Ligand unit Pc specifically binds to EGFR and c-Met.

[0073] In some embodiments, the ligand unit Pc that specifically binds to EGFR and c-Met comprises at least a bispecific antibody; preferably, the bispecific antibody is at least bivalent; preferably, the bispecific antibody comprises two different Fc regions, and the Fc comprises a mutation that promotes heterodimerization, such as a Knob-into-Hole mutation.

[0074] In some embodiments, the Ligand unit Pc comprises a first, a second, a third, and a fourth polypeptide chain:

[0075] (1) The first polypeptide chain comprises an antibody heavy chain variable region domain targeting EGFR, wherein the first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO.11;

[0076] (2) the second polypeptide chain comprises an antibody heavy chain variable region domain targeting c-Met, and the first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO. 12;

[0077] (3) the third polypeptide chain comprises an antibody light chain variable region domain targeting EGFR, and the first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO. 13; and

[0078] (4) The fourth polypeptide chain comprises an antibody light chain variable region domain targeting c-Met, and the first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO.14.

[0079] In some embodiments, n is a real number selected from 1-8, for example, n is 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.9 .6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0; preferably, n is selected from a real number of 2-8, more preferably, n is selected from a real number of 6-8.

[0080] In a second aspect, the present disclosure provides a linker unit represented by formula (L), which is used to connect a ligand unit and a drug unit to obtain a ligand-drug conjugate.

[0081] Among them, X 1 、X 2 、X 3 、X 4 、X 5 , L a , L b , L c , n1 and n2 are as defined in any embodiment of the first aspect above, the a-terminus of L is connected to the Ligand unit, and the b-terminus of L is connected to the Drug unit.

[0082] In a third aspect, the present disclosure provides a drug-linker compound represented by formula (II) or a pharmaceutically acceptable salt thereof,

[0083] Among them, X 1 、X 2 、X 3 、X 4 、X 5 , L a , L b , L c , n1, n2 and drug unit D are as defined in any one of the first aspects above;

[0084] X is selected from absent, O or NR e1 ;

[0085] R 1 Selected from N(R e2 )(R e3), C1-C 10 Alkyl, C3-C 10 Cycloalkyl, 4-14 membered heterocyclic group, C6-C 10 Aryl or 5-10 membered heteroaryl; the C1-C 10 Alkyl, C3-C 10 Cycloalkyl, 4-14 membered heterocyclic group, C6-C 10 Aryl or 5-10 membered heteroaryl is optionally substituted with one or more R f Replace, each R f Independently selected from halogen, CN, =O, C1-C3 alkyl, OH, O(C1-C3 alkyl), NH2, NH(C1-C3 alkyl) or N(C1-C3 alkyl)2;

[0086] R e1 、R e2 、R e3 Each is independently selected from H, C1-C6 alkyl, C3-C6 cycloalkyl or 4-7 membered heterocyclyl.

[0087] In some embodiments, X is selected from O.

[0088] In some embodiments, R 1 Selected from C1-C6 alkyl or C3-C6 cycloalkyl, the C1-C6 alkyl or C3-C6 cycloalkyl is optionally substituted by one or more R f replace.

[0089] In some embodiments, R 1 is selected from methyl, tert-butyl or cyclopentyl.

[0090] In some embodiments, the drug-linker compound represented by formula (II) or a pharmaceutically acceptable salt thereof is selected from the following compounds or pharmaceutically acceptable salts thereof:

[0091] On the other hand, the present disclosure provides a pharmaceutical composition comprising the aforementioned general formula Pc-(LD) n ligand-drug conjugate or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

[0092] In another aspect, the present disclosure provides a method for treating mammalian tumors, comprising administering to a mammal, preferably a human, in need of such treatment a therapeutically effective amount of the aforementioned compound of the general formula Pc-(LD) n A ligand-drug conjugate or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0093] On the other hand, the present disclosure provides the aforementioned general formula Pc-(LD) n Use of a ligand-drug conjugate or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof in preparing a drug for treating tumors.

[0094] On the other hand, the present disclosure provides the aforementioned general formula Pc-(LD) n Use of a ligand-drug conjugate or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof in treating tumors.

[0095] On the other hand, the present disclosure provides the aforementioned general formula Pc-(LD) for treating tumors n A ligand-drug conjugate or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0096] In another aspect, the present disclosure provides a compound of the formula Pc-(LD) n A method for preparing a ligand-drug conjugate or a pharmaceutically acceptable salt thereof comprises the step of coupling the drug-linker compound of the general formula (II) disclosed above with the aforementioned ligand, optionally, the ligand is an antibody or an antigen-binding fragment thereof.

[0097] In another aspect, the present disclosure provides a compound of the formula Pc-(LD) n A method for preparing a ligand-drug conjugate or a pharmaceutically acceptable salt thereof, comprising the step of connecting the aforementioned drug unit D of the present disclosure to the aforementioned ligand unit Pc; optionally, the aforementioned drug unit D is connected to the aforementioned ligand unit Pc through the aforementioned linker unit L; optionally, the ligand unit Pc is an antibody or an antigen-binding fragment thereof.

[0098] The ligand-drug conjugates provided by the present disclosure have significant anti-tumor activity and / or reduced toxic side effects and / or improved product uniformity and / or higher stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0099] FIG1 shows the inhibition of tumor growth in an HCC827 subcutaneous tumor model by exemplary EGFR-cMet ADCs of the present disclosure and a control ADC.

[0100] FIG2 shows the effects of exemplary EGFR-cMet ADCs of the present disclosure and a control ADC on mouse body weight in an HCC827 subcutaneous tumor model.

[0101] FIG3 shows the inhibition of tumor growth by exemplary EGFR-cMet ADCs of the present disclosure and a control ADC in an HCT116 subcutaneous tumor model.

[0102] FIG4 shows the effects of exemplary EGFR-cMet ADCs of the present disclosure and a control ADC on mouse body weight in an HCT116 subcutaneous tumor model.

[0103] FIG5 shows the inhibition of tumor growth in a PC9-6 subcutaneous tumor model by exemplary EGFR-cMet ADCs of the present disclosure and a control ADC.

[0104] FIG6 shows the effects of exemplary EGFR-cMet ADCs of the present disclosure and a control ADC on mouse body weight in a PC9-6 subcutaneous tumor model.

[0105] FIG7 shows the inhibition of tumor growth in a PA-1 subcutaneous tumor model by exemplary CDH-6 ADCs of the present disclosure and a control ADC.

[0106] Definitions and Explanations of Terms

[0107] Unless otherwise indicated, the terms used in this disclosure have the following meanings. The definitions of groups and terms described in this disclosure, including their definitions as examples, exemplary definitions, preferred definitions, definitions described in tables, and definitions of specific compounds in the examples, may be combined and coupled with each other in any manner. A particular term should not be considered as undefined or unclear unless specifically defined, but should be understood according to its ordinary meaning in the art. When a trade name appears in this document, it is intended to refer to the corresponding commercial product or its active ingredient.

[0108] The term "ligand" refers to a macromolecular compound that can recognize and bind to an antigen or receptor associated with a target cell. The role of the ligand is to present the drug to the target cell population bound to the ligand. These ligands include but are not limited to protein hormones, lectins, growth factors, antibodies or other molecules that can bind to cells. In the embodiments of the present disclosure, the ligand or ligand unit is represented by Pc, and the ligand can form a connection bond with the linker unit through a heteroatom on the ligand. In some embodiments of the present disclosure, the ligand unit Pc may include a heteroatom group (such as an S atom or an N atom) on the ligand for connection to the linker unit, or may not include the above-mentioned heteroatom group for connection to the linker unit. In some embodiments of the present disclosure, the ligand is selected from an antibody or an antigen-binding fragment, and the antibody is selected from a chimeric antibody, a humanized antibody, a fully human antibody or a mouse antibody; in some embodiments of the present disclosure, the antibody is a monoclonal antibody.

[0109] The term "linker" or "linker unit" refers to a chemical structure fragment or chemical bond that is connected to the ligand at one end and to the drug at the other end.

[0110] The term "drug" refers to a small molecule compound that has biological activity in vivo. In some embodiments of the present disclosure, drugs include glucocorticoid receptor agonists with anti-inflammatory properties, cytotoxic drugs that kill or inhibit cell growth, and protein degraders that promote the degradation of target proteins within cells. In some embodiments of the present disclosure, protein degraders are small molecule protein degraders.

[0111] The term "ligand-drug conjugate" refers to a ligand linked to a biologically active drug via a stable linker unit. In some embodiments of the present disclosure, the "ligand-drug conjugate" is an antibody-drug conjugate (ADC), which is a monoclonal antibody or antibody fragment linked to a biologically active drug via a stable linker unit.

[0112] The term "DAR" or "drug-to-antibody ratio" refers to the average number of small molecule glucocorticoid receptor agonist drugs attached to each antibody molecule. In the antibody-drug conjugates of the present disclosure, DAR is defined by the variable "n," which can be either an integer or a decimal.

[0113] The term "antibody" is used in the broadest sense to refer to a polypeptide or combination of polypeptides that contains sufficient sequence from the variable region of an immunoglobulin heavy chain and / or sufficient sequence from the variable region of an immunoglobulin light chain to be able to specifically bind to an antigen. "Antibodies" herein encompass various forms and structures, as long as they exhibit the desired antigen binding activity. "Antibodies" herein include alternative protein scaffolds or artificial scaffolds with transplanted complementary determining regions (CDRs) or CDR derivatives. Such scaffolds include antibody-derived scaffolds (which contain mutations introduced to, for example, stabilize the three-dimensional structure of the antibody) and fully synthetic scaffolds containing, for example, biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1): 121-129 (2003); Roque et al., Biotechnol. Prog. 20: 639-654 (2004). Such scaffolds may also include non-antibody derived scaffolds, such as scaffold proteins known in the art that can be used to graft CDRs, including but not limited to tenascin, fibronectin, peptide aptamers, and the like.

[0114] The "antibody" herein includes a typical "tetrabodies," which are immunoglobulins composed of two heavy chains (HC) and two light chains (LC); the heavy chain refers to a polypeptide chain that, in the N-terminal to C-terminal direction, consists of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain; and, when the full-length antibody is of the IgE isotype, optionally also includes a heavy chain constant region CH4 domain; the light chain is a polypeptide chain that, in the N-terminal to C-terminal direction, consists of a light chain variable region (VL) and a light chain constant region (CL); the heavy chains are linked to each other and to each other by disulfide bonds, forming a "Y"-shaped structure. Due to the different amino acid composition and arrangement order of the constant regions of the heavy chains of immunoglobulins, their antigenicity is also different. Based on this, the "immunoglobulins" referred to herein can be divided into five classes, or isotypes, namely IgM, IgD, IgG, IgA, and IgE. Their corresponding heavy chains are μ, δ, γ, α, and ε, respectively. Igs of the same class are further divided into subclasses based on the amino acid composition of their hinge regions and the number and location of disulfide bonds in their heavy chains. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA can be divided into IgA1 and IgA2. Light chains are classified as either kappa or lambda chains based on differences in their constant regions. Each of the five Ig classes can have either kappa or lambda chains.

[0115] The "antibodies" herein also include antibodies that do not contain light chains, for example, heavy-chain antibodies (HCAbs) produced by dromedary camels (Camelus dromedarius), Bactrian camels (Camelus bactrianus), llamas (Lama glama), guanacos (Lama guanicoe) and alpacas (Vicugna pacos), as well as immunoglobulin new antigen receptors (Ig new antigen receptor, IgNAR) found in cartilaginous fish such as sharks.

[0116] The "antibodies" herein may be derived from any animal, including but not limited to humans and non-human animals, which may be selected from primates, mammals, rodents and vertebrates, such as camelids, llamas, guanacos, alpacas, sheep, rabbits, mice, rats or cartilaginous fish (e.g., sharks).

[0117] "Antibodies" herein include, but are not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), monovalent antibodies, multivalent antibodies, whole antibodies, fragments of whole antibodies, naked antibodies, conjugated antibodies, chimeric antibodies, humanized antibodies, or fully human antibodies.

[0118] The term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, that is, except for possible variants (e.g., containing naturally occurring mutations or generated during the production of the preparation, such variants typically being present in small amounts), the individual antibodies comprising the population are identical and / or bind to the same epitope. In contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on the antigen. The modifier "monoclonal" herein should not be interpreted as requiring the antibody or antigen-binding molecule to be produced by any particular method. For example, monoclonal antibodies can be made by a variety of techniques, including but not limited to hybridoma technology, recombinant DNA methods, phage library display technology, and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus and other methods known in the art.

[0119] The term "natural antibody" refers to an antibody produced and paired by the immune system of a multicellular organism. The term "engineered antibody" herein refers to a non-natural antibody obtained by techniques such as genetic engineering and antibody engineering. Exemplarily, "engineered antibodies" include humanized antibodies, antibody fragments (e.g., scFv, sdAb, etc.), bispecific antibodies, etc.

[0120] The term "HER2," also known as "human epidermal growth factor receptor 2" or ErbB2, is a cellular oncogene and a transmembrane glycoprotein with tyrosine kinase activity that activates intracellular tyrosine kinase-regulated signal transduction systems. HER2 gene copy number amplification and protein overexpression lead to overactivation of signaling, a biological function closely associated with the development, malignant proliferation, invasion and metastasis, and resistance to chemotherapy and radiotherapy in various cancers. Both the oncogene and its protein product (P185) are overexpressed and amplified in a variety of tumors. Positive expression of HER2 has been observed in various cancers, including breast, ovarian, gastric, esophageal, salivary gland, lung, bile duct, bladder, prostate, and colorectal cancers.

[0121] The term "CDH6" herein is also known as "cadherin 6", which refers to a cell adhesion molecule, which is a member of the cadherin family of cell-cell adhesion molecules. CDH6 is a single transmembrane protein consisting of 790 amino acids, which is classified as a type II cadherin family, and the protein has an N-terminal extracellular and C-terminal intracellular domain. The CDH6 protein used in the present disclosure can be directly purified from cells expressing CDH6 of humans or non-human mammals (e.g., rats, mice, or monkeys), and can be used subsequently, or the cell membrane fraction of the above-mentioned cells can be prepared and can be used as CDH6 protein. Alternatively, CDH6 can also be obtained by allowing host cells to produce CDH6 by in vitro synthesis or by genetic manipulation. According to this genetic manipulation, CDH6 protein can be obtained, specifically, CDH6 cDNA is incorporated into a vector capable of expressing CDH6 cDNA, and then CDH6 is synthesized in a solution containing enzymes, substrates, and energy materials required for transcription and translation, or by transforming host cells of other prokaryotes or eukaryotes, thereby allowing them to express CDH6. CDH6-expressing cells or CDH6-expressing cell lines based on the above genetic manipulations can also be used to present CDH6 protein. Alternatively, an expression vector incorporating CDH6 cDNA can be directly administered to an animal to be immunized, and CDH6 can be expressed in the animal thus immunized.

[0122] The term "epidermal growth factor receptor" or "EGFR" herein refers to human EGFR (also known as HER1 or ErbB1) having the amino acid sequence shown in GenBank Accession No. NP_005219, and naturally occurring variants thereof.

[0123] The term "hepatocyte growth factor receptor" or "c-Met" herein refers to human c-Met having the amino acid sequence shown in GenBank Accession No. NP_001120972 and its natural variants.

[0124] The term "monospecific" is intended to mean having one or more binding sites, wherein each binding site binds to the same epitope of the same antigen.

[0125] The term "multispecific antibody" refers to an antibody having at least two antigen-binding sites, each of which binds to a different epitope of the same antigen or to different epitopes of different antigens. Thus, terms such as "bispecific," "trispecific," and "tetraspecific" refer to the number of different epitopes to which an antibody / antigen-binding molecule can bind.

[0126] The term "valent" refers to the presence of a specified number of binding sites in an antibody / antigen-binding molecule. Thus, the terms "monovalent," "divalent," "tetravalent," and "hexavalent" refer to the presence of one, two, four, and six binding sites, respectively, in an antibody / antigen-binding molecule.

[0127] "Full-length antibody," "intact antibody," and "intact antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to that of a native antibody.

[0128] "Antigen-binding fragment" and "antibody fragment" herein are used interchangeably herein and do not have the entire structure of an intact antibody, but only contain a portion or partial variant of an intact antibody that has the ability to bind to an antigen. "Antigen-binding fragment" or "antibody fragment" herein includes, but is not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, VHH, and scFv.

[0129] Papain digestion of intact antibodies generates two identical antigen-binding fragments, called "Fab" fragments, each containing the variable domains of the heavy and light chains, as well as the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Thus, the term "Fab fragment" herein refers to an antibody fragment comprising a light chain fragment comprising the VL domain and constant domain (CL) of the light chain, and a VH domain and first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine ​​residues of the constant domain carry a free thiol group. Pepsin treatment produces a F(ab')2 fragment having two antigen-binding sites (two Fab fragments) and a portion of the Fc region.

[0130] "Fv fragment" is the smallest fragment produced by IgG and IgM that contains a complete antigen binding site. The Fv fragment has the same binding properties and similar three-dimensional binding properties as Fab. The VH and VL chains of the Fv fragment are bound together by non-covalent interactions.

[0131] The term "scFv" (single-chain variable fragment) refers to a single polypeptide chain comprising a VL and VH domain, wherein the VL and VH are connected by a linker (see, e.g., Bird et al., Science 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers useful in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond can also exist between the VH and VL of the scFv to form a disulfide-linked Fv (dsFv).

[0132] The term "diabody" refers to a protein whose VH and VL domains are expressed on a single polypeptide chain, but with a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and create two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak RJ et al., Structure 2:1121-1123 (1994)).

[0133] The term "chimeric antibody" refers to an antibody in which a portion of its light chain and / or heavy chain is derived from one antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and another portion of the light chain and / or heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but in any case, it still retains binding activity to the target antigen (USP 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)). For example, the term "chimeric antibody" may include antibodies (e.g., human-mouse chimeric antibodies) in which the heavy and light chain variable regions of the antibody are derived from a first antibody (e.g., a murine antibody), and the heavy and light chain constant regions of the antibody are derived from a second antibody (e.g., a human antibody).

[0134] The term "humanized antibody" refers to a non-human antibody that has been genetically engineered and whose amino acid sequence has been modified to increase homology with the sequence of a human antibody. Generally speaking, all or part of the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., FR and / or constant region in the variable region) comes from a human immunoglobulin (recipient antibody). Humanized antibodies generally retain or partially retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, ability to increase immune cell activity, ability to enhance immune response, etc.

[0135] The term "fully human antibody" refers to an antibody having a variable region in which both FR and CDR are derived from human germline immunoglobulin sequences. In addition, if the antibody comprises a constant region, the constant region is also derived from human germline immunoglobulin sequences. "fully human antibody" herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, "fully human antibody" herein does not include antibodies in which the CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been transplanted to human framework sequences.

[0136] The term "variable region" refers to the region of an antibody heavy or light chain that binds to the antigen. "Heavy chain variable region" is used interchangeably with "VH" and "HCVR," and "light chain variable region" is used interchangeably with "VL" and "LCVR." The variable domains of the heavy and light chains of native antibodies (VH and VL, respectively) generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. The terms "complementarity determining region" and "CDR" are used interchangeably herein and generally refer to the hypervariable region (HVR) of the heavy chain variable region (VH) or light chain variable region (VL). This region is also called the complementarity determining region because it can form precise complementarity with the antigen epitope in terms of spatial structure. The heavy chain variable region CDR can be abbreviated as HCDR, and the light chain variable region CDR can be abbreviated as LCDR. The terms "framework region" or "FR region" are used interchangeably herein and refer to those amino acid residues in the heavy chain variable region or light chain variable region of an antibody other than the CDR. Typically, a typical antibody variable region consists of four FR regions and three CDR regions in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0137] The "CDRs" herein can be annotated and defined by methods known in the art, including but not limited to the Kabat numbering system, the Chothia numbering system, or the IMGT numbering system, and the tool websites used include but are not limited to the AbRSA website (http: / / cao.labshare.cn / AbRSA / cdrs.php), the abYsis website (www.abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi), and the IMGT website (http: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi#results). The CDRs herein include overlaps and subsets of amino acid residues defined in different ways.

[0138] The term "heavy chain constant region" herein refers to the carboxyl-terminal portion of an antibody heavy chain, which is not directly involved in antibody-antigen binding but exhibits effector functions, such as interactions with Fc receptors. It has a more conserved amino acid sequence than the variable domains of antibodies. A "heavy chain constant region" comprises at least: a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, or variants or fragments thereof. "Heavy chain constant regions" include "full-length heavy chain constant regions" and "heavy chain constant region fragments." The former has a structure substantially similar to that of a native antibody constant region, while the latter only comprises "a portion of a full-length heavy chain constant region." For example, a typical "full-length antibody heavy chain constant region" consists of a CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is an IgE, it also includes a CH4 domain; when the antibody is a heavy chain antibody, it does not include a CH1 domain. For example, a typical "heavy chain constant region fragment" can be selected from the CH1, Fc, or CH3 domains.

[0139] The term "light chain constant region" herein refers to the carboxyl terminal portion of the antibody light chain, which is not directly involved in binding the antibody to the antigen, and the light chain constant region can be selected from a constant kappa domain or a constant lambda domain.

[0140] The term "Fc" herein refers to the antibody carboxyl terminal portion formed by papain hydrolysis of intact antibodies, typically comprising the CH3 and CH2 domains of an antibody. The Fc region includes, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary slightly, the Fc region of a human IgG heavy chain is typically defined as extending from the amino acid residue at Cys226 position or from Pro230 to its carboxyl terminal. The C-terminal lysine (residue 447 according to the Kabat numbering system) in the Fc region can, for example, be present in the production or purification process of an antibody, or by removing the nucleic acid recombinant engineering of an encoding antibody heavy chain. Therefore, the Fc region may include or may not include Lys447.

[0141] The term "identity" herein can be calculated in the following manner: to determine the percentage of "identity" between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment, or non-homologous sequences can be discarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position.

[0142] The percent identity between the two sequences will vary depending on the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0143] Mathematical algorithms can be used to compare sequences and calculate percent identity between two sequences. For example, the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm, which has been integrated into the GAP program in the GCG software package (available at www.gcg.com), is used with a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6 to determine the percent identity between two amino acid sequences. For another example, the GAP program in the GCG software package (available at www.gcg.com) is used with a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6 to determine the percent identity between two nucleotide sequences. A particularly preferred parameter set (and the one that should be used unless otherwise stated) is the Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0144] Herein, “n is a real number of 1-8” means that n is any real number greater than or equal to 1 and less than or equal to 8.

[0145] As used herein, "an integer from x to y" includes both every integer in the interval (including x and y) and a range with any two of the integers as endpoints. For example, "m4 is an integer selected from 1 to 10" means that m4 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and also includes m4 selected from a range with any two of the 10 integers as endpoints, for example, m4 is selected from 2 to 9, 3 to 9, 3 to 8, 4 to 6, etc.

[0146] In this article Indicates the attachment site.

[0147] The diagrammatic representations of racemates or enantiomerically pure compounds herein are from Maehr, J. Chem. Ed. 1985, 62: 114-120. Unless otherwise indicated, wedge and dotted wedge keys are used. To indicate the absolute configuration of a stereocenter, use black real and imaginary bonds. Indicates the relative configuration of a stereocenter (such as the cis-trans configuration of an alicyclic compound).

[0148] The term "tautomer" refers to functional group isomers resulting from the rapid shift of an atom in a molecule between two positions. The compounds of the present disclosure may exhibit tautomerism. Tautomeric compounds may exist as two or more interconvertible species. Tautomers generally exist in equilibrium, and attempts to isolate a single tautomer usually result in a mixture whose physical and chemical properties are consistent with a mixture of compounds. The position of equilibrium depends on the chemical properties within the molecule. For example, in many aliphatic aldehydes and ketones such as acetaldehyde, the keto form predominates, while in phenols, the enol form predominates. The present disclosure encompasses all tautomeric forms of the compounds.

[0149] The term "stereoisomer" refers to isomers resulting from different spatial arrangements of atoms in a molecule, including cis-trans isomers, enantiomers and diastereomers.

[0150] The compounds of the present invention may have asymmetric atoms such as carbon atoms, sulfur atoms, nitrogen atoms, phosphorus atoms or asymmetric double bonds, so that the compounds of the present invention may exist in specific geometric or stereoisomeric forms. Specific geometric or stereoisomeric forms may be cis and trans isomers, E and Z geometric isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic mixtures or other mixtures thereof, such as mixtures enriched in enantiomers or diastereomers, all of which are within the definition of the compounds of the present invention and mixtures thereof. Additional asymmetric carbon atoms, asymmetric sulfur atoms, asymmetric nitrogen atoms or asymmetric phosphorus atoms may be present in substituents such as alkyl groups, and all of which are within the definition of the compounds of the present invention and mixtures thereof. Compounds of the present disclosure containing an asymmetric atom can be isolated in optically pure or racemic forms. Optically pure forms can be resolved from racemic mixtures or synthesized by using chiral starting materials or reagents.

[0151] The term "substituted" refers to the replacement of any one or more hydrogen atoms on a particular atom by a substituent, as long as the valence state of the particular atom is normal and the substituted compound is stable. When the substituent is an oxo (i.e., =O), it means that two hydrogen atoms are replaced. Oxo does not occur on aromatic groups.

[0152] The term "optional" or "optionally" refers to that the event or situation described subsequently may or may not occur, and the description includes that the event or situation occurs and that the event or situation does not occur. For example, an ethyl group is "optionally" substituted with halogen, meaning that the ethyl group may be unsubstituted (CH2CH3), monosubstituted (CH2CH2F, CH2CH2Cl, etc.), polysubstituted (CHFCH2F, CH2CHF2, CHFCH2Cl, CH2CHCl2, etc.), or fully substituted (CF2CF3, CF2CCl3, CCl2CCl3, etc.). It will be appreciated by those skilled in the art that for any group comprising one or more substituents, any sterically impossible and / or incomposable replacement or substitution pattern will not be introduced.

[0153] When any variable (such as R a 、R b ) appears more than once in the composition or structure of a compound, its definition is independent in each case. For example, if a group is represented by two R b is replaced, then each R b There are independent options.

[0154] When the number of a linking group is 0, such as -(CH2)0-, it means that the linking group is a bond.

[0155] When one of the variables is selected from a chemical bond or does not exist, it means that the two groups it connects are directly connected. For example, when L in ALZ represents a bond, it means that the structure is actually AZ.

[0156] When the linking group mentioned in this article does not specify its connection direction, its connection direction is arbitrary. 3 -W 4 -W 5 W in 4 Selected from "[A-(CR d1 R d2 ) m2 -NR d3 ] m3 -B", at this time W 4 You can connect W from left to right 3 and W 5 Constitute "W 3 -[A-(CR d1 R d2 ) m2 -NR d3 ] m3 -BW 5 ", you can also connect W from right to left 3 and W 5 Constitute "W 3-B-[NR d3 -(CR d1 R d2 ) m2 -A] m3 -W 5 ”.

[0157] In this article, C m -C n , means having an integer number of carbon atoms in the range of mn.

[0158] The term "alkyl" refers to a group of the formula C n H 2n+1 The term "C1-C 10 "Alkyl" refers to a straight or branched saturated hydrocarbon group having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms. Specific examples of the alkyl group include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl-, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl or 1,2-dimethylbutyl; The term "C1-C6 alkyl" refers to a straight chain or branched saturated hydrocarbon group having 1, 2, 3, 4, 5 or 6 carbon atoms. The "C1-C6 alkyl" herein refers to a straight chain or branched saturated hydrocarbon group having 1, 2, 3, 4, 5 or 6 carbon atoms. 10 The term "alkyl" may include "C1-C6 alkyl", "C1-C4 alkyl" or "C1-C3 alkyl" and the like, and the "C1-C6 alkyl" may further include "C1-C4 alkyl" or "C1-C3 alkyl".

[0159] The term "alkylene" refers to a saturated divalent hydrocarbon group having a straight or branched chain. 10 "Alkylene" refers to a straight or branched saturated divalent hydrocarbon group having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms, including but not limited to methylene, CH2CH2 or CH2CH2CH2, etc.

[0160] The term "cycloalkyl" refers to a fully saturated monovalent carbon ring that exists in the form of a monocyclic, fused, bridged, or spirocyclic ring. The term "C3-C6 cycloalkyl" should be understood to mean a saturated monocyclic, fused, spirocyclic, or bridged ring having 3, 4, 5, or 6 carbon atoms. Specific examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0161] The term "C3-C6 cycloalkylene" refers to a fully saturated divalent carbon ring having 3, 4, 5 or 6 ring carbon atoms and existing in the form of a monocyclic, fused, bridged or spirocyclic ring.

[0162] The term "heterocyclyl" refers to a fully saturated or partially saturated monovalent group in the form of a monocyclic, fused, spirocyclic or bridged ring, wherein the ring atoms contain 1-5 heteroatoms or heteroatomic groups (i.e., heteroatomic groups containing heteroatoms), wherein the "heteroatoms or heteroatomic groups" include, but are not limited to, nitrogen atom (N), oxygen atom (O), sulfur atom (S), phosphorus atom (P), boron atom (B), -S(=O)2-, -S(=O)-, -P(=O)2-, -P(=O)-, -NH-, -S(=O)(=NH)-, -C(=O)NH- or -NHC(=O)NH-. The term "4-7 membered heterocyclyl" refers to a heterocyclyl group having 4, 5, 6 or 7 ring atoms, wherein the ring atoms contain 1-3 heteroatoms or heteroatomic groups independently selected from the above-mentioned heteroatoms or heteroatomic groups. Examples of 4-membered heterocyclic groups include, but are not limited to, azetidinyl and oxetanyl; examples of 5-membered heterocyclic groups include, but are not limited to, tetrahydrofuranyl, dioxolyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrrolinyl, 4,5-dihydrooxazole, or 2,5-dihydro-1H-pyrrolyl; examples of 6-membered heterocyclic groups include, but are not limited to, tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl, trithianyl, tetrahydropyridinyl, or 4H-[1,3,4]thiadiazinyl; examples of 7-membered heterocyclic groups include, but are not limited to, diazepanyl. Preferably, "4-7-membered heterocyclic group" may include "4-7-membered heterocycloalkyl," "5-6-membered heterocyclic group," "5-6-membered heterocycloalkyl," and the like.

[0163] The term "heterocyclylene" refers to a fully saturated or partially saturated divalent group in the form of a monocyclic, fused, spirocyclic or bridged ring, wherein the ring atoms contain 1-5 heteroatoms or heteroatom groups, wherein the "heteroatoms or heteroatom groups" include but are not limited to nitrogen atom (N), oxygen atom (O), sulfur atom (S), phosphorus atom (P), boron atom (B), -S(=O)2-, -S(=O)-, -P(=O)2-, -P(=O)-, -NH-, -S(=O)(=NH)-, -C(=O)NH- or -NHC(=O)NH-, etc.

[0164] The term "C2-C3 alkenyl" refers to a straight or branched unsaturated hydrocarbon group containing one double bond and having 2 or 3 carbon atoms, specific examples of which include but are not limited to vinyl, allyl, (E)-2-methylvinyl, and (Z)-2-methylvinyl.

[0165] The term "C2-C3 alkynyl" refers to a straight or branched unsaturated hydrocarbon group containing one triple bond and having 2 or 3 carbon atoms, specific examples of which include but are not limited to ethynyl (-C≡CH), prop-1-ynyl (-C≡CCH3), and prop-2-ynyl (-CH2C≡CH).

[0166] The term "halo" or "halogen" refers to fluorine, chlorine, bromine and iodine.

[0167] The term "hydrophilic group" refers to a group that can dissolve or ionize in water and is easily water-affinity. Hydrophilic groups generally contain one or more O, N, P, S, OH, NH, NH2, C=O, S(=O)2, P(=O)2, and any combination thereof, such as polyether, polyhydroxy, polyamine, polysaccharide, peptide, carboxylic acid, sulfonic acid, phosphoric acid, and other structural units.

[0168] The term "treatment" refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) undesirable physiological changes or pathological changes in the treated subject, such as the progression of cancer, autoimmune diseases and viral infections. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, weakening of the disease extent, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial relief or complete relief), whether detectable or undetectable. Objects in need of treatment include objects already suffering from a disease or disease, objects susceptible to a disease or disease, or objects intended to prevent a disease or disease. When referring to terms such as slowing down, alleviating, weakening, alleviating, alleviating, etc., their meanings also include situations such as elimination, disappearance, and non-occurrence.

[0169] The term "effective amount" refers to an amount of a therapeutic agent that, when administered alone or in combination with another therapeutic agent to a cell, tissue, or subject, is effective in preventing or ameliorating a disease symptom or the progression of that disease. "Effective amount" also refers to an amount of a compound sufficient to alleviate symptoms, e.g., to treat, cure, prevent, or alleviate a related medical condition, or to increase the rate of treatment, cure, prevention, or alleviation of such a condition. When an active ingredient is administered alone to a subject, a therapeutically effective dose refers to that ingredient alone. When a combination is used, a therapeutically effective dose refers to the combined amounts of the active ingredients that produce a therapeutic effect, whether administered in combination, sequentially, or simultaneously.

[0170] The term "subject" refers to an organism that is being treated for a particular disease or condition as described herein. Examples of subjects and patients include mammals, such as humans, primates (e.g., monkeys), or non-primate mammals, being treated for a disease or condition.

[0171] The amount of a compound of the disclosure that constitutes a "therapeutically effective amount" varies depending on the compound, the disease state and its severity, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by one skilled in the art based on their own knowledge and this disclosure.

[0172] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0173] The term "pharmaceutically acceptable salt" refers to a salt of a pharmaceutically acceptable acid or base, including a salt formed between a compound and an inorganic acid or organic acid, and a salt formed between a compound and an inorganic base or an organic base.

[0174] The term "pharmaceutical composition" refers to a mixture of one or more compounds of the present disclosure or their salts and a pharmaceutically acceptable excipient. The purpose of a pharmaceutical composition is to facilitate administration of the compounds of the present disclosure to an organism.

[0175] The term "pharmaceutically acceptable excipient" refers to an excipient that is non-irritating to organisms and does not impair the biological activity and properties of the active compound. Suitable excipients are well known to those skilled in the art and include, for example, carbohydrates, waxes, water-soluble and / or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, and the like.

[0176] The word "comprise" or "comprises" and its English variations such as comprises or comprising are to be understood as having an open and non-exclusive meaning, ie, "including but not limited to".

[0177] The present disclosure also includes isotopically labeled compounds of the present disclosure that are identical to those described herein, but where one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into the compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as 2 H. 3 H. 11 C. 13 C. 14 C. 13 N. 15 N. 15 O. 17 O. 18 O. 31 P. 32 P. 35 S.18 F. 123 I. 125 I and 36 Cl et al.

[0178] Certain isotopically labeled compounds of the present disclosure (e.g., 3 H and 14 C-labeled) can be used in compound and / or substrate tissue distribution assays. 3 H) and carbon-14 (i.e. 14 C) isotopes are particularly preferred due to their ease of preparation and detectability. Positron emitting isotopes, such as 15 O. 13 N. 11 C and 18 F can be used in positron emission tomography (PET) studies to determine substrate occupancy. Isotopically labeled compounds of the disclosure can generally be prepared by following procedures analogous to those disclosed in the Schemes and / or Examples below, by substituting an isotopically labeled reagent for a non-isotopically labeled reagent.

[0179] The pharmaceutical compositions of the present disclosure may be suitable for parenteral administration, such as sterile solutions, suspensions, or lyophilized products in suitable unit dosage forms. For example, the pharmaceutical compositions of the present disclosure may be in the form of sterile aqueous injection solutions for intramuscular or subcutaneous administration. The pharmaceutical compositions of the present disclosure may be administered in other solvents or media, such as water, Ringer's solution, or isotonic sodium chloride solution.

[0180] In all administration methods of the compounds described herein, the daily dosage is 0.001 mg / kg to 600 mg / kg body weight, preferably 0.05 mg / kg to 200 mg / kg body weight, more preferably 0.1 mg / kg to 100 mg / kg body weight, in single or divided doses.

[0181] The compounds disclosed herein can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining the same with other chemical synthesis methods, and equivalent substitutions well known to those skilled in the art. Preferred embodiments include, but are not limited to, the examples disclosed herein.

[0182] The chemical reactions of the embodiments of the present disclosure are carried out in a suitable solvent that is compatible with the chemical transformations of the present disclosure and the reagents and materials required. In order to obtain the compounds of the present disclosure, it is sometimes necessary for those skilled in the art to modify or select synthetic steps or reaction schemes based on existing embodiments.

[0183] An important consideration in synthetic route planning in the art is the selection of appropriate protecting groups for reactive functional groups (e.g., amino and carboxyl groups in the present disclosure). For example, reference may be made to Greene's Protective Groups in Organic Synthesis (4th Ed). Hoboken, New Jersey: John Wiley & Sons, Inc. All references cited in the present disclosure are hereby incorporated into the present disclosure in their entirety.

[0184] This disclosure uses the following abbreviations: Example

[0185] The present disclosure is described in detail below by way of examples, but is not intended to limit the present disclosure in any way. While the present disclosure has been described in detail herein, including specific embodiments thereof, it will be apparent to those skilled in the art that various modifications can be made to the specific embodiments without departing from the spirit and scope of the present disclosure. All reagents used in the present disclosure are commercially available and used without further purification.

[0186] Unless otherwise specified, the ratios expressed for mixed solvents are volume ratios.

[0187] Unless otherwise stated, % refers to wt%.

[0188] Compounds are manually or Software naming, commercially available compounds use supplier catalog names.

[0189] The structures of the compounds were determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). The units of NMR shifts are 10 -6 (ppm). The solvents for NMR determination are deuterated dimethyl sulfoxide, deuterated chloroform, deuterated methanol, etc., and the internal standard is tetramethylsilane (TMS); "IC 50 " refers to the half-maximal inhibitory concentration, which is the concentration at which half of the maximum inhibitory effect is achieved. 50 "half maximal effect concentration" refers to the concentration that causes 50% of the maximum effect.

[0190] Example 1, tert-butyl 3-(2-methylsulfonyl-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propanoate (Compound 1)

[0191] Step 1: Synthesis of tert-butyl 2-methylthio-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (Intermediate 1-1)

[0192] To a mixture of tert-butyl 2,4-dioxopiperidine-1-carboxylate (15 g) and 150 mL of toluene at room temperature under a nitrogen atmosphere was added N,N-dimethylformamide dimethyl acetal (DMF-DMA) (9.2 g). The resulting mixture was stirred at 25°C for 1 hour. After the solvent was removed under reduced pressure, methylisothiourea hemisulfate (10.8 g) was added. Potassium carbonate (14.7 g) and anhydrous ethanol (200 mL) were added, and the resulting mixture was stirred at 25°C for 4 hours. The solvent was then removed under reduced pressure, and the crude title compound was separated by column chromatography (EA / PE, 0–30%) to obtain the crude product, which was used directly in the next step.

[0193] LC-MS: m / z(ESI):296.1[M+H] + .

[0194] Step 2: Synthesis of 2-methylthio-7,8-dihydropyrido[4,3-d]pyrimidin-5(6H)-one (Intermediate 1-2)

[0195] The intermediate 1-1 obtained in the previous step was dissolved in 100 mL of dichloromethane, and 25 mL of trifluoroacetic acid was added. The mixture was stirred at 25°C for 2 h. The solvent was then removed under reduced pressure, and 100 mL of ethyl acetate was added to slurry. The mixture was filtered and dried to obtain the title compound (10.0 g).

[0196] LC-MS: m / z(ESI):196.1[M+H] + .

[0197] Step 3: Synthesis of tert-butyl 3-(2-methylthio-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propanoate (Intermediate 1-3)

[0198] To a mixture of intermediate 1-2 (9.75 g) and THF (240 mL) was added NaH (5 g, 60% by mass in mineral oil) at 0°C. The resulting mixture was reacted at 0°C for 30 min. Tert-butyl 3-bromopropionate (15.68 g) was then added to the reaction mixture, and the resulting mixture was heated to 70°C for 2 h. Afterward, 2 mL of water was added to the mixture, and the solvent was removed under reduced pressure. The title compound (9.75 g) was obtained by column chromatography (EA / PE, 0–50%).

[0199] LC-MS: m / z(ESI):324.4[M+H] + .

[0200] Step 4: Synthesis of tert-butyl 3-(2-methylsulfonyl-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propanoate (Compound 1)

[0201] To a mixture of intermediate 1-3 (5 g) and DCM (150 mL) was added mCPBA (12.6 g, 85% purity) at 0°C, and the resulting mixture was reacted at room temperature for 30 min. The mixture was then decompressed, and the residue was separated by column chromatography (EA / PE, 0–100%) to afford the title compound (5.2 g).

[0202] LC-MS: m / z(ESI):356.4[M+H] + .

[0203] 1 H NMR (400MHz, DMSO-d6) δ9.24 (s, 1H), 3.76 (t, J = 6.7Hz, 2H), 3.70 (t, J = 7.0Hz ,2H),3.43(s,3H),3.24(t,J=6.7Hz,2H),2.57(t,J=7.0Hz,2H),1.39(s,9H).

[0204] Example 2, tert-butyl 3-(2-methylsulfonyl-5-oxo-5,7-dihydro-6H-pyrrolo[3,4-d]pyrimidin-6-yl)propanoate (Compound 2)

[0205] Step 1: Synthesis of ethyl 4-(((tert-Butoxycarbonyl)amino)methyl)-2-methylthiopyrimidine-5-carboxylate (Intermediate 2-1)

[0206] To a mixture of ethyl 4-chloro-2-methylthiopyrimidine-5-carboxylate (5.82 g), potassium [(tert-butoxycarbonylamino)methyl]trifluoroborate (4.74 g), cesium carbonate (13.0 g), palladium acetate (448 mg), and n-butyldi(1-adamantyl)phosphine (1.43 g) was added 1,4-dioxane (100 mL) and water (10 mL) at room temperature under a nitrogen atmosphere. The resulting mixture was stirred at 90°C for 6 h. The solvent was then removed under reduced pressure, and the title compound (4147 mg) was obtained by column chromatography (EA / PE, 0–50%).

[0207] LC-MS: m / z(ESI):328.1[M+H] + .

[0208] Step 2: Synthesis of (5-ethoxycarbonyl-2-methylthiopyrimidin-4-yl)methylamine hydrochloride (Intermediate 2-2)

[0209] To a solution of intermediate 2-1 (1637 mg) in 10 mL of ethyl acetate was added 4 M hydrogen chloride in ethyl acetate (10 mL) at room temperature under nitrogen atmosphere. The resulting mixture was stirred at room temperature for 4 h. The mixture was then filtered and the filter cake was washed with ethyl acetate to obtain the title compound (1231 mg).

[0210] LC-MS: m / z(ESI):228.3[M+H] + .

[0211] Step 3: Synthesis of tert-butyl 3-(2-methylthio-5-oxo-5,7-dihydro-6H-pyrrolo[3,4-d]pyrimidin-6-yl)propanoate (Intermediate 2-3)

[0212] Under a nitrogen atmosphere, a mixture of intermediate 2-2 (500 mg), cesium carbonate (2.14 g), tert-butyl 3-bromopropionate (916 mg), and xylene (10 mL) was heated to 80°C for 16 h. The solvent was then removed under reduced pressure, and the title compound (28 mg) was obtained by column chromatography (EA / PE, 0-30%) and thin-layer chromatography (EA / PE = 1 / 1).

[0213] LC-MS: m / z(ESI):310.0[M+H] + .

[0214] Step 4: Synthesis of tert-butyl 3-(2-methylsulfonyl-5-oxo-5,7-dihydro-6H-pyrrolo[3,4-d]pyrimidin-6-yl)propanoate (Compound 2)

[0215] Meta-chloroperbenzoic acid (mCPBA) (78.74 mg, 85% purity) was added to a solution of intermediate 2-3 (30 mg) in 1 mL of dichloromethane at 0°C, and the resulting mixture was stirred at room temperature for 1 hour. The mixture was then separated by column chromatography (EA / PE, 0–66%) to afford the title compound (14.0 mg).

[0216] LC-MS: m / z(ESI):342.2[M+H] + .

[0217] 1 H NMR (400MHz, DMSO-d6) δ = 9.37 (s, 1H), 4.76 (s, 2H), 3.78 (t, J = 7.1Hz, 2H), 3.48 (s, 3H), 2.64 (t, J = 7.1Hz, 2H), 1.39 (s, 9H)

[0218] Example 3, tert-butyl 3-(2-ethanesulfonyl-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propanoate (Compound 3)

[0219] Step 1: Synthesis of tert-butyl 3-(2-ethylthio-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propanoate (Intermediate 3-1)

[0220] To a mixture of compound 1 (50 mg) and ethanethiol (180 mg) was added dichloromethane (1 mL) and 2M sodium hydroxide solution (0.5 mL) at room temperature. The resulting mixture was stirred at 25°C for 3 h. The solvent was then removed under reduced pressure, and the title compound (45 mg) was obtained by column chromatography (THF / PE, 0-30%).

[0221] LC-MS: m / z(ESI):338.1[M+H] + .

[0222] Step 2: Synthesis of tert-butyl 3-(2-ethanesulfonyl-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propanoate (Compound 3)

[0223] m-Chloroperbenzoic acid (92.05 mg) was added to a solution of intermediate 3-1 (45 mg) in dichloromethane (2 mL) at 0°C, and the resulting mixture was stirred at room temperature for 1 h. The mixture was then separated by column chromatography (EA / PE, 0–100%) to obtain the title compound (15.6 mg).

[0224] LC-MS: m / z(ESI):370.1[M+H] + .

[0225] 1 H NMR (400MHz, DMSO-d6) δ = 9.25 (s, 1H), 3.77 (t, J = 6.7Hz, 2H), 3.71 (t, J = 7.0Hz, 2H), 3.62 (q, J =7.4Hz,2H),3.25(t,J=6.7Hz,2H),2.57(t,J=7.0Hz,2H),1.40(s,9H),1.26(t,J=7.3Hz,3H)

[0226] Example 4, tert-butyl 3-(2-butanesulfonyl-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propanoate (Compound 4)

[0227] Step 1: Synthesis of tert-butyl 3-(2-butylthio-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propanoate (Intermediate 4-1)

[0228] To a mixture of compound 1 (50 mg) and 1-butanethiol (19 mg) was added dichloromethane (1 mL) and 2M sodium hydroxide solution (0.5 mL) at room temperature. The resulting mixture was stirred at 25°C for 3 h. The solvent was then removed under reduced pressure, and the title compound (47 mg) was obtained by column chromatography (THF / PE, 0-18%).

[0229] LC-MS: m / z(ESI):366.4[M+H] + .

[0230] Step 2: Synthesis of tert-butyl 3-(2-butanesulfonyl-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propanoate (Compound 4)

[0231] At 0°C, m-chloroperbenzoic acid (88.76 mg) was added to a solution of intermediate 4-1 (47 mg) in dichloromethane (2 mL), and the resulting mixture was stirred at room temperature for 1 h. The mixture was then separated by column chromatography (EA / PE, 0-100%) to obtain the title compound (10.7 mg).

[0232] LC-MS: m / z(ESI):398.1[M+H] +

[0233] 1 H NMR (400MHz, DMSO-d6) δ = 9.25 (s, 1H), 3.77 (t, J = 6.7Hz, 2H), 3.71 (t, J = 7.0Hz, 2H), 3.63-3.58 (m, 2H), 3.25 (t ,J=6.7Hz,2H),2.57(t,J=7.0Hz,2H),1.72-1.63(m,2H),1.45-1.41(m,2H),1.40(s,9H),0.89(t,J=7.3Hz,3H)

[0234] Example 5, tert-butyl 3-(2-isobutanesulfonyl-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propanoate (Compound 5)

[0235] Step 1: Synthesis of tert-butyl 3-(2-isobutylthio-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propanoate (Intermediate 5-1)

[0236] To a mixture of compound 1 (50 mg) and 1-butanethiol (250 mg) was added dichloromethane (1 mL) and 2 M sodium hydroxide solution (0.5 mL) at room temperature. The resulting mixture was stirred at 25°C for 3 h. The solvent was then removed under reduced pressure, and the title compound (47 mg) was obtained by column chromatography (THF / PE, 0-18%).

[0237] LC-MS: m / z(ESI):366.4[M+H] + .

[0238] Step 2: Synthesis of tert-butyl 3-(2-isobutanesulfonyl-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propanoate (Compound 5)

[0239] At 0°C, m-chloroperbenzoic acid (88.76 mg) was added to a solution of intermediate 5-2 (47 mg) in dichloromethane (2 mL), and the resulting mixture was stirred at room temperature for 1 h. The mixture was then separated by column chromatography (EA / PE, 0-100%) to obtain the title compound (7.6 mg).

[0240] LC-MS: m / z(ESI):398.1[M+H] +

[0241] 1 H NMR (400MHz, DMSO-d6) δ = 9.25 (s, 1H), 3.77 (t, J = 6.7Hz, 2H), 3.71 (t, J = 7.0Hz, 2H), 3.54 (d, J = 6.6Hz, 2H), 3 .25(t,J=6.7Hz,2H),2.57(t,J=7.0Hz,2H),2.21(t,J=6.7,13.4Hz,1H),1.40(s,9H),1.04(d,J=6.7Hz,6H)

[0242] Example 6, tert-butyl 3-(2-isopropylsulfonyl-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propanoate (Compound 6)

[0243] Step 1: Synthesis of tert-butyl 3-(2-isopropylthio-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propanoate (Intermediate 6-1)

[0244] To a mixture of compound 1 (50 mg) and isopropyl mercaptan (500 mg) was added dichloromethane (1 mL) and 2M sodium hydroxide solution (0.5 mL) at room temperature. The resulting mixture was stirred at 25°C for 3 h. The solvent was then removed under reduced pressure, and the title compound (45 mg) was obtained by column chromatography (THF / PE, 0-30%).

[0245] LC-MS: m / z(ESI):352.2[M+H] + .

[0246] Step 2: Synthesis of tert-butyl 3-(2-isopropylsulfonyl-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propanoate (Compound 6)

[0247] At 0°C, m-chloroperbenzoic acid (88 mg) was added to a solution of intermediate 6-1 (45 mg) in dichloromethane (2 mL), and the resulting mixture was stirred at room temperature for 1 h. The mixture was then separated by column chromatography (EA / PE, 0-100%) to obtain the title compound (18.5 mg).

[0248] LC-MS: m / z(ESI):384.1[M+H] + .

[0249] 1 H NMR (400MHz, DMSO-d6) δ = 9.25 (s, 1H), 3.99–3.94 (m, 1H), 3.77 (t, J = 6.7Hz, 2H), 3.71 (t, J = 7. 0Hz,2H),3.25(t,J=6.7Hz,2H),2.57(t,J=7.0Hz,2H),1.40(s,9H),1.28(s,3H),1.27(s,3H)

[0250] Example 7, tert-Butyl 3-(2-cyclopentanesulfonyl-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propanoate (Compound 7)

[0251] Step 1: Synthesis of tert-butyl 3-(2-cyclopentylthio-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propanoate (Intermediate 7-1)

[0252] To a solution of compound 1 (1.066 g) in DCM (10 mL) were added cyclopentylmercaptan (367 mg) and 2 M sodium hydroxide solution (1.65 mL) at room temperature. The resulting mixture was stirred at room temperature for 30 minutes. The mixture was then separated by column chromatography (EA / PE, 0–50%) to obtain the crude title compound, which was used directly in the next reaction.

[0253] LC-MS: m / z(ESI):378.4[M+H] + .

[0254] Step 2: Synthesis of tert-butyl 3-(2-cyclopentanesulfonyl-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propanoate (Compound 7)

[0255] To a mixture of the crude compound from the previous step and DCM (50 mL) was added mCPBA (2.43 g, 85% purity) at 0°C. The resulting mixture was reacted at room temperature for 30 min. The mixture was then concentrated under reduced pressure, and the residue was separated by column chromatography (EA / PE, 0–100%) to afford the title compound (890 mg).

[0256] LC-MS: m / z(ESI):410.5[M+H] + .

[0257] 1 H NMR(400MHz, DMSO-d6)δ9.23(s,1H),4.30–4.17(m,1H),3.76(t,J=6.7Hz,2H),3.70(t,J=7.0Hz,2H ),3.24(t,J=6.7Hz,2H),2.56(t,J=7.0Hz,2H),2.04–1.85(m,4H),1.74–1.57(m,4H),1.39(s,9H).

[0258] Example 8, tert-Butyl 3-(2-tert-butylsulfonyl-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propanoate (Compound 8)

[0259] Step 1: Synthesis of tert-butyl 3-(2-tert-butylthio-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propanoate (Intermediate 8-1)

[0260] To a solution of compound 1 (1.066 g) in DCM (10 mL) was added tert-butyl mercaptan (324 mg) and 2 M sodium hydroxide solution (1.65 mL) sequentially at room temperature. The resulting mixture was stirred vigorously at room temperature for 30 minutes. The mixture was then separated by column chromatography (EA / PE, 0–50%) to obtain the crude title compound, which was used directly in the next reaction.

[0261] LC-MS: m / z(ESI):366.4[M+H] + .

[0262] Step 2: Synthesis of tert-butyl 3-(2-tert-butylsulfonyl-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propanoate (Compound 8)

[0263] To a mixture of the crude compound from the previous step and DCM (50 mL) was added mCPBA (2.43 g, 85% purity) at 0°C. The resulting mixture was reacted at room temperature for 30 minutes. The mixture was then concentrated under reduced pressure, and the residue was separated by column chromatography (EA / PE, 0–100%) to afford the title compound (831 mg).

[0264] LC-MS: m / z(ESI):398.4[M+H] + .

[0265] 1 H NMR (400MHz, DMSO-d6) δ9.25 (s, 1H), 3.76 (t, J = 6.7Hz, 2H), 3.70 (t, J = 7.0Hz ,2H),3.23(t,J=6.7Hz,2H),2.57(t,J=7.0Hz,2H),1.40(s,9H),1.37(s,9H).

[0266] Example 9. Synthesis of tert-butyl 3-(2-(1-adamantylsulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propanoate (Compound 9)

[0267] Step 1: Synthesis of tert-butyl 3-(2-(1-adamantylthio)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propanoate (Intermediate 9-1)

[0268] To a solution of compound 1 (410 mg) in DCM (10 mL) at room temperature were added 2M aqueous NaOH (0.692 mL) and 1-adamantanethiol (291 mg), and the resulting mixture was stirred at 25°C for 2 h. The reaction mixture was then diluted with water (5 mL) and extracted with DCM (3 mL x 3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and separated by column chromatography (THF / PE, 0–60%) to afford the title compound (351 mg).

[0269] LC-MS: m / z(ESI):444.0[M+H] +

[0270] Step 2: Synthesis of tert-butyl 3-(2-(1-adamantylsulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propanoate (Compound 9)

[0271] mCPBA (546 mg, 85% purity) was added to a solution of intermediate 9-1 (351 mg) in DCM (5 mL) at room temperature and stirred at 25°C for 2 hours. The solvent was then concentrated under reduced pressure, and the mixture was separated by column chromatography (THF / PE, 0-50%) to obtain the title compound (300 mg). This was then purified by HPLC (column: C18 150 × 30 mm × 5 μm; mobile phase: [Phase A: water (0.225% formic acid), Phase B: acetonitrile]; B%: 43%-63%, 11 min) to obtain the title compound (24.7 mg).

[0272] LC-MS: m / z(ESI):476.1[M+H] +

[0273] 1 H NMR (400MHz, DMSO-d6) δ = 9.26 (s, 1H), 3.77-3.75 (m, 2H), 3.71-3.69 (m, 2H), 3.24 (t, J = 6.7 Hz,2H),2.60-2.55(m,2H),2.08(s,3H),2.00-1.92(m,6H),1.68-1.58(m,6H),1.41(s,9H).

[0274] Example 10, tert-Butyl 3-(2-(4-methoxyphenylsulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propanoate (Compound 10)

[0275] Step 1: Synthesis of tert-butyl 3-(2-(4-methoxyphenylthio)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propanoate (Intermediate 10-1)

[0276] To a mixture of compound 1 (40.0 mg), 4-methoxythiophenol (23.7 mg), and sodium hydroxide (4.50 mg) was added dichloromethane (2 mL) and water (56 μL) at room temperature under a nitrogen atmosphere. The resulting mixture was stirred at room temperature for 1 hour. The solvent was then removed under reduced pressure, and the title compound (40.0 mg) was obtained by column chromatography (EA / PE, 0-80%).

[0277] LC-MS: m / z(ESI):416.3[M+H] + .

[0278] Step 2: Synthesis of tert-butyl 3-(2-(4-methoxyphenylsulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propanoate (Compound 10)

[0279] At 0°C, mCPBA (117 mg, 85% purity) was added to a solution of intermediate 10-1 (40 mg) in 1 mL of dichloromethane. The resulting mixture was stirred at room temperature for 1 hour. The mixture was then separated by column chromatography (THF / PE, 0-80%) and purified by HPLC (column: C18 150 × 30 mm × 5 μm; mobile phase: [Phase A: water (0.225% formic acid), Phase B: acetonitrile]; B%: 45%-75%, 9 min) to obtain the title compound (5.0 mg).

[0280] LC-MS: m / z(ESI):448.2[M+H] + .

[0281] 1 H NMR (400MHz, DMSO-d6) δ9.14(s,1H),7.93(d,J=8.8Hz,2H),7.20(d,J=8.8Hz,2H),3. 87(s,3H),3.73–3.64(m,4H),3.18(t,J=6.7Hz,2H),2.55-2.51(m,2H),1.38(s,9H).

[0282] Example 11, tert-Butyl 4-((6-(3-tert-butoxy-3-oxopropyl)-5-oxo-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-2-yl)sulfonyl)piperidine-1-carboxylate (Compound 11)

[0283] Step 1: Synthesis of tert-butyl 4-((6-(3-tert-butoxy-3-oxopropyl)-5-oxo-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-2-yl)thio)piperidine-1-carboxylate (Intermediate 11-1)

[0284] To a mixture of compound 1 (50 mg) and tert-butyl 4-mercaptopiperidine-1-carboxylate (30.57 mg) was added dichloromethane (1 mL) and 2 M sodium hydroxide solution (0.5 mL) at room temperature. The resulting mixture was stirred at 25°C for 3 h. The solvent was then removed under reduced pressure, and the mixture was separated by column chromatography (THF / PE, 0-30%) to obtain the title compound (50 mg).

[0285] LC-MS: m / z(ESI):515.1[M+Na] + .

[0286] Step 2: Synthesis of tert-butyl 4-((6-(3-tert-butoxy-3-oxopropyl)-5-oxo-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-2-yl)sulfonyl)piperidine-1-carboxylate (Compound 11)

[0287] mCPBA (87.57 mg) was added to a solution of intermediate 11-1 (50 mg) in dichloromethane (1 mL) at 0°C, and the resulting mixture was stirred at room temperature for 1 h. The mixture was then separated by column chromatography (EA / PE, 0–100%) to obtain the title compound (20.7 mg).

[0288] LC-MS: m / z(ESI):547.2[M+Na] + .

[0289] 1 H NMR (400MHz, DMSO-d6) δ=9.25(s,1H),4.08-3.96(m,3H),3.77(t,J=6.7Hz,2H),3.73-3.66(m,2H),3.25(t,J=6.8H z,2H),2.92-2.75(m,2H),2.61-2.55(m,2H),1.95(d,J=12.1Hz,2H),1.58-1.46(m,2H),1.41(s,9H),1.40(s,9H).

[0290] Example 12, tert-Butyl 3-(2-(4,4-difluorocyclohexylsulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propanoate (Compound 12)

[0291] Step 1: Synthesis of 4,4-difluorocyclohexanol methanesulfonate (Intermediate 12-1)

[0292] To a solution of 4,4-difluorocyclohexanol (1 g) and triethylamine (1.23 mL) in DCM (36 mL) was added methanesulfonyl chloride (1.01 g) dropwise at 0°C, and the resulting mixture was stirred at 25°C for 16 h. After the reaction, the mixture was poured into water (50 mL) and extracted with ethyl acetate (20 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude title compound (1.57 g), which was used directly in the next reaction without further purification.

[0293] 1 H NMR (400MHz, DMSO-d6) δ = 4.93-4.77 (m, 1H), 3.22 (s, 3H), 2.11-1.80 (m, 8H)

[0294] Step 2: Synthesis of S-(4,4-difluorocyclohexylthiol acetate (Intermediate 12-2)

[0295] To a solution of intermediate 12-1 (785 mg) in DMA (18 mL) was added potassium thioacetate (1.26 g), and the resulting mixture was heated at 80°C for 18 h. The mixture was then cooled to room temperature, and ethyl acetate (50 mL) and water (50 mL) were added. The mixture was extracted with ethyl acetate (3 × 20 mL). The combined organic phases were dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (THF / PE, 0-6%) to give the title compound (368 mg).

[0296] 1 H NMR (400MHz, DMSO-d6) δ = 3.58-3.46 (m, 1H), 2.32 (s, 3H), 2.08-1.85 (m, 8H).

[0297] Step 3: Synthesis of tert-butyl 3-(2-(4,4-difluorocyclohexylthio)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propanoate (Intermediate 12-3)

[0298] To a solution of intermediate 12-2 (100 mg) in THF (1 mL) was added NaOH (2 M, 1.54 mL), and the reaction mixture was stirred at 25°C for 2 hours. Compound 1 (90 mg) was then added, and the mixture was stirred at 25°C for 1 hour. The mixture was then poured into 5 mL of water and extracted with ethyl acetate (2 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, concentrated under reduced pressure, and separated by column chromatography (EA / PE, 0–31%) to obtain the title compound (80 mg).

[0299] LC-MS: m / z(ESI):428.1[M+H] +

[0300] Step 4: Synthesis of tert-butyl 3-(2-(4,4-difluorocyclohexylsulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propanoate (Compound 12)

[0301] mCPBA (151.96 mg, 85% purity) was added to a solution of Intermediate 12-3 (80 mg) in DCM (1 mL) and stirred at 25°C for 2 h. The mixture was then poured into 5 mL of water and extracted with ethyl acetate (2 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was separated by column chromatography (EA / PE, 0-40%) and purified by preparative thin-layer chromatography (PE / EA = 1 / 2) to obtain the title compound (9.3 mg).

[0302] LC-MS: m / z(ESI):460.2[M+H] +

[0303] 1 H NMR (400MHz, DMSO-d6) δ=9.25(s,1H),4.05–3.95(m,1H),3.82-3.73(m,2H),3.70(t,J=6.8Hz,2H),3.24(t,J=6.5Hz,2H),2.64-2.54(m,2H),2.19 -1.93(m,6H),1.71(d,J=12.4Hz,2H),1.39(s,9H).

[0304] 19 F NMR (376MHz, DMSO-d6) δ = -91.16 (d, J = 235.6Hz), -100.27 (d, J = 235.6Hz).

[0305] Example 13, tert-Butyl 3-(2-methanesulfonyl-5-oxo-8,8-dimethyl-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propanoate (Compound 13)

[0306] Step 1: Synthesis of tert-butyl 3-(2-methylthio-5-oxo-8,8-dimethyl-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propanoate (Intermediate 13-1)

[0307] To a mixture of intermediate 1-3 (4.75 g) and THF (100 mL) was added NaH (1.76 g, 60% by mass in mineral oil) at 0°C. The resulting mixture was reacted at 0°C for 30 min. Methyl iodide (6.24 g) and DMF (20 mL) were then added to the resulting mixture, and the reaction continued at room temperature for 4 h. 2 mL of water was then added to the mixture, and the solvent was removed under reduced pressure. The title compound (2.77 g) was obtained by column chromatography (EA / PE, 0–50%).

[0308] LC-MS: m / z(ESI):352.4[M+H] + .

[0309] Step 2: Synthesis of tert-butyl 3-(2-methylthio-5-oxo-8,8-dimethyl-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propanoate (Compound 13)

[0310] To a mixture of the crude compound from the previous step and DCM (100 mL) was added mCPBA (6.38 g, 85% purity) at 0°C. The resulting mixture was allowed to react at room temperature for 30 minutes. The mixture was then concentrated under reduced pressure, and the residue was separated by column chromatography (EA / PE, 0–100%) to obtain the title compound (2.87 g).

[0311] LC-MS: m / z(ESI):384.4[M+H] + .

[0312] 1 H NMR (400MHz, DMSO-d6) δ9.27(s,1H),3.70(t,J=7.0Hz,2H),3.60(s,2H),3.45(s,3H),2.58(t,J=7.0Hz,2H),1.39(s,9H),1.33(s,6H).

[0313] Example 14, tert-butyl 3-(2-tert-butylsulfonyl-5-oxo-8,8-dimethyl-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propanoate (Compound 14)

[0314] Step 1: Synthesis of tert-butyl 3-(2-tert-butylthio-5-oxo-8,8-dimethyl-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propanoate (Intermediate 14-1)

[0315] To a solution of compound 13 (600 mg) in DCM (10 mL) were added tert-butyl mercaptan (212 mg) and 2M sodium hydroxide solution (0.94 mL) at room temperature. The resulting mixture was stirred at room temperature for 30 minutes. The mixture was then separated by column chromatography (EA / PE, 0–50%) to obtain the crude title compound, which was used directly in the next reaction.

[0316] LC-MS: m / z(ESI):394.4[M+H] + .

[0317] Step 2: Synthesis of tert-butyl 3-(2-tert-butylsulfonyl-5-oxo-8,8-dimethyl-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propanoate (Compound 14)

[0318] To a mixture of the crude compound from the previous step and DCM (30 mL) was added mCPBA (1.27 g, 85% purity) at 0°C. The resulting mixture was reacted at room temperature for 30 min. The mixture was then concentrated under reduced pressure, and the residue was separated by column chromatography (EA / PE, 0–100%) to afford the title compound.

[0319] LC-MS: m / z(ESI):426.5[M+H] + .

[0320] Example 15, tert-butyl 3-(2-tert-butylsulfonyl-5-oxopyrimido[5,4-c]quinolin-6(5H)-yl)propanoate (Compound 15)

[0321] Step 1: Synthesis of 2-methylthiopyrimido[5,4-c]quinolin-5(6H)-one (Intermediate 15-1)

[0322] Under a nitrogen atmosphere, THF (40 mL) and water (6 mL) were added to a mixture of ethyl 2-methylthio-4-chloropyrimidine-5-carboxylate (1.86 g), 2-aminophenylboronic acid (1.096 g), Pd(dppf)Cl2 (585 mg), and Cs2CO3 (5.2 g). The resulting mixture was stirred at 70°C for 4 h. The mixture was then cooled to room temperature overnight and filtered. The filter cake was washed with water and petroleum ether to obtain the title compound (400 mg).

[0323] LC-MS: m / z(ESI):244.3[M+H] + .

[0324] Step 2: Synthesis of tert-butyl 3-(2-methylthio-5-oxopyrimido[5,4-c]quinolin-6(5H)-yl)propanoate (Intermediate 15-2)

[0325] Under a nitrogen atmosphere, to a mixture of intermediate 15-1 (90 mg) dissolved in DMF (3 mL) and THF (3 mL) was added NaH (59 mg, 60% by mass in mineral oil). The resulting mixture was stirred at room temperature for 30 minutes. Tert-butyl 3-bromopropionate (309 mg) was then added, and the resulting mixture was reacted at 70°C for 16 hours. 0.5 mL of water was then added to the mixture, and the solvent was removed under reduced pressure. The crude title compound was then separated by column chromatography and used directly in the next reaction.

[0326] LC-MS: m / z(ESI):372.4[M+H] + .

[0327] Step 3: Synthesis of tert-butyl 3-(2-methylsulfonyl-5-oxopyrimido[5,4-c]quinolin-6(5H)-yl)propanoate (Intermediate 15-3)

[0328] To a mixture of the crude compound from the previous step and DCM (10 mL) was added mCPBA (200 mg, 85% purity) at 0°C. The resulting mixture was reacted at room temperature for 30 min. The mixture was then concentrated under reduced pressure, and the residue was separated by column chromatography (EA / PE, 0–100%) to afford the crude title compound, which was used directly in the next reaction.

[0329] LC-MS: m / z(ESI):404.4[M+H] + .

[0330] Step 4: Synthesis of tert-butyl 3-(2-tert-butylthio-5-oxopyrimido[5,4-c]quinolin-6(5H)-yl)propanoate (Intermediate 15-4)

[0331] To a solution of the crude compound from the previous step in DCM (10 mL) was added tert-butyl mercaptan (200 mg) and 2M sodium hydroxide solution (0.5 mL) sequentially at room temperature. The resulting mixture was stirred vigorously at room temperature for 30 minutes. The mixture was then separated by column chromatography (EA / PE, 0–50%) to obtain the crude title compound.

[0332] LC-MS: m / z(ESI):414.4[M+H] + .

[0333] Step 5: Synthesis of tert-butyl 3-(2-tert-butylsulfonyl-5-oxopyrimido[5,4-c]quinolin-6(5H)-yl)propanoate (Compound 15)

[0334] To a mixture of the crude compound from the previous step and DCM (10 mL) was added mCPBA (200 mg, 85% purity) at 0°C. The resulting mixture was reacted at room temperature for 30 min. The mixture was then concentrated under reduced pressure, and the residue was separated by column chromatography (EA / PE, 0–100%) to afford the title compound (28.9 mg).

[0335] LC-MS: m / z(ESI):446.5[M+H] + .

[0336] 1 H NMR(400MHz,DMSO-d6)δ9.78(s,1H),8.73–8.67(m,1H),7.96–7.89(m,1H),7.81(d,J=8.6Hz ,1H),7.55(t,J=7.5Hz,1H),4.62–4.55(m,2H),2.70–2.62(m,2H),1.45(s,9H),1.38(s,9H).

[0337] Example 16, 3-(2-tert-butylsulfonyl-5-oxo-5,7,8,9-tetrahydro-6H-pyrimido[5,4-c]azepine -6-yl)tert-butyl propionate (Compound 16)

[0338] Step 1: Synthesis of 2-methylthio-7,8-dihydroquinolin-5(6H)-one (Intermediate 16-1)

[0339] A mixture of 1,3-cyclohexanedione (11.2 g) and DMF-DMA (35.7 g) was stirred at room temperature under nitrogen for 1 hour, then heated to 50°C for 1 hour. The solvent was removed under reduced pressure, followed by the addition of methylisothiourea hemisulfate (13.9 g), sodium carbonate (12.7 g), and 1,4-dioxane (200 mL). The resulting mixture was stirred at 100°C for 16 hours. The solvent was then removed under reduced pressure, and the title compound (5 g) was obtained by column chromatography (EA / PE, 0–30%).

[0340] LC-MS: m / z(ESI):195.4[M+H] + .

[0341] Step 2: 2-Methylthio-6,7,8,9-tetrahydro-5H-pyrimido[5,4-c]azepine Synthesis of -5-ketone (Intermediate 16-2)

[0342] To a solution of Intermediate 16-1 (1.94 g) in 20 mL of trifluoroacetic acid was added trimethylsilylazide (2.3 g) at 0°C under a nitrogen atmosphere. The resulting mixture was stirred at 25°C for 10 days. The mixture was then poured into 200 mL of water and extracted with dichloromethane (50 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate and separated by column chromatography (EA / PE, 0–100%) to obtain the title compound (210 mg).

[0343] LC-MS: m / z(ESI):210.5[M+H] + .

[0344] Step 3: 3-(2-methylthio-5-oxo-5,7,8,9-tetrahydro-6H-pyrimido[5,4-c]azepine Synthesis of tert-butyl butyl propionate (intermediate 16-3)

[0345] To a mixture of intermediate 16-2 (210 mg) and THF (5 mL) was added NaH (80 mg, 60% by mass in mineral oil) at 0°C. The resulting mixture was reacted at 0°C for 30 minutes. Then, tert-butyl 3-bromopropionate (418 mg) was added to the resulting mixture, and the mixture was heated to 70°C and reacted for 16 hours. The solvent was then removed under reduced pressure, and the crude title compound was separated by column chromatography (EA / PE, 0–70%) to obtain the crude product, which was used directly in the next step.

[0346] LC-MS: m / z(ESI):338.4[M+H] + .

[0347] Step 4: 3-(2-methanesulfonyl-5-oxo-5,7,8,9-tetrahydro-6H-pyrimido[5,4-c]azepine Synthesis of tert-butyl butyl propionate (intermediate 16-4)

[0348] To a mixture of the crude compound from the previous step and DCM (5 mL) was added mCPBA (200 mg) at 0°C, and the resulting mixture was reacted at room temperature for 30 min. The mixture was then separated by column chromatography (EA / PE, 0–100%) to afford the crude title compound, which was used directly in the next reaction.

[0349] LC-MS: m / z(ESI):370.4[M+H] + .

[0350] Step 5: 3-(2-tert-Butylthio-5-oxo-5,7,8,9-tetrahydro-6H-pyrimido[5,4-c]azepine Synthesis of tert-butyl butyl propionate (intermediate 16-5)

[0351] To a mixture of the crude compound from the previous step and DCM (5 mL) was added tert-butyl mercaptan (0.5 mL) and 2 M sodium hydroxide solution (1 mL) with vigorous stirring. The resulting mixture was allowed to react at room temperature for 1 h. The mixture was then separated by column chromatography (EA / PE, 0–100%) to afford the crude title compound, which was used directly in the next reaction.

[0352] LC-MS: m / z(ESI):380.4[M+H] + .

[0353] Step 6: 3-(2-tert-Butylsulfonyl-5-oxo-5,7,8,9-tetrahydro-6H-pyrimido[5,4-c]azepine Synthesis of tert-butyl (6-amino)propanoate (Compound 16): To a mixture of the crude compound obtained in the previous step and DCM (5 mL) was added mCPBA (200 mg) at 0°C. The resulting mixture was reacted at room temperature for 30 minutes. The mixture was then separated by column chromatography (EA / PE, 0–100%) to obtain the title compound (14.2 mg).

[0354] LC-MS: m / z(ESI):412.5[M+H] + .

[0355] 1 H NMR (400MHz, DMSO-d6) δ9.04 (s, 1H), 3.75 (t, J = 7.0Hz, 2H), 3.37–3.34 (m, 2H), 2.99 ( t,J=7.3Hz,2H),2.61(t,J=7.0Hz,2H),2.21–2.11(m,2H),1.42(s,9H),1.38(s,9H).

[0356] Example 17, tert-Butyl 3-(2-(cyclopentanesulfonyl)-5-oxo-8,8-di(2,5,8,11-tetraoxatridecan-13-yl)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propanoate (Compound 17)

[0357] Step 1: Synthesis of tert-butyl 3-(2-cyclopentylthio-5-oxo-8,8-di(2,5,8,11-tetraoxatridecan-13-yl)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propanoate (Intermediate 17-1)

[0358] Under a nitrogen atmosphere at 0°C, intermediate 7-1 (50 mg) was dissolved in DMF (1 mL). NaH (26.5 mg, 60% active ingredient) was added. The reaction was stirred at 25°C for 30 min, and 13-bromo-2,5,8,11-tetraoxatridecane (179.57 mg) was added. The reaction mixture was stirred at 25°C under nitrogen for 3 hours. The mixture was then poured into 30 mL of water and extracted with ethyl acetate (20 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, concentrated under reduced pressure, and separated by column chromatography (THF / PE, 0-100%) to obtain the title compound (25 mg).

[0359] LC-MS: m / z(ESI):758.5[M+H] +

[0360] Step 2: Synthesis of tert-butyl 3-(2-cyclopentanesulfonyl-5-oxo-8,8-di(2,5,8,11-tetraoxatridecan-13-yl)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propanoate (Compound 17)

[0361] Intermediate 17-1 (25 mg) was dissolved in anhydrous dichloromethane (1 mL) at 0°C, and m-chloroperbenzoic acid (22.77 mg, 85% purity) was added. The reaction mixture was stirred at 25°C for 1 hour. The mixture was then purified by reverse-phase HPLC (column: Boston Green ODS 150*30mm*5um; mobile phase: [Phase A: water (0.225% formic acid), Phase B: acetonitrile]; B%: 50%-80%, 10 min) to obtain the title compound (2.2 mg).

[0362] LC-MS: m / z(ESI):790.4[M+H] + .

[0363] 1 H NMR (400MHz, D2O) δ = 9.28 (s, 1H), 4.36-4.24 (m, 1H), 3.84 (t, J = 6.3Hz, 2H), 3.80 (s, 2H), 3.63-3.49 (m, 20H), 3.47-3.36 (m, 8H) ),3.32(s,6H),2.67(t,J=6.3Hz,2H),2.33-2.22(m,2H),2.20-2.10(m,2H),2.09-1.97(m,4H),1.70–1.62(m,4H),1.39(s,9H)

[0364] Example 18, tert-Butyl 6-(2-(cyclopentanesulfonyl)-5-oxo-8,8-di(2,5,8,11-tetraoxatridecan-13-yl)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)hexanoate (Compound 18)

[0365] Step 1: Synthesis of tert-butyl 6-(2-methylthio-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)hexanoate (Intermediate 18-1)

[0366] To a mixture of intermediate 1-2 (1.0 g) and DMF (20 mL) at 0°C under a nitrogen atmosphere was added NaH (307.28 mg, 60% by mass in mineral oil). The reaction was stirred at 25°C for 30 minutes, followed by the addition of tert-butyl 6-bromohexanoate (1.93 g). The reaction mixture was stirred at 25°C under nitrogen for 1 hour. The mixture was then poured into 50 mL of water and extracted with ethyl acetate (30 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, concentrated under reduced pressure, and separated by column chromatography (THF / PE, 0-50%) to afford the title compound (500 mg).

[0367] LC-MS: m / z(ESI):366.2[M+H] + .

[0368] Step 2: Synthesis of tert-butyl 6-(2-methanesulfonyl-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)hexanoate (Intermediate 18-2)

[0369] Intermediate 18-1 (500 mg) was dissolved in anhydrous dichloromethane (10 mL) at 0°C, and m-chloroperbenzoic acid (944.30 mg, 85% purity) was added. The reaction mixture was stirred at 25°C for 1 h. The mixture was then separated by column chromatography (THF / PE, 0–50%) to obtain the title compound (500 mg).

[0370] LC-MS: m / z(ESI):342.0[M-56] + .

[0371] Step 3: Synthesis of tert-butyl 6-(2-cyclopentylthio-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)hexanoate (Intermediate 18-3)

[0372] Intermediate 18-2 (500 mg) and cyclopentanethiol (193 mg) were dissolved in dichloromethane (10 mL) and 2M sodium hydroxide solution (5 mL) at room temperature. The resulting mixture was stirred at 25°C for 3 h. The solvent was then removed under reduced pressure, and the title compound (460 mg) was obtained by column chromatography (THF / PE, 0-30%).

[0373] LC-MS: m / z(ESI):442.1[M+Na] + .

[0374] Step 4: Synthesis of tert-butyl 6-(2-(cyclopentylthio)-5-oxo-8,8-di(2,5,8,11-tetraoxatridecan-13-yl)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)hexanoate (Intermediate 18-4)

[0375] Under a nitrogen atmosphere at 0°C, intermediate 18-3 (350 mg) was dissolved in DMF (5 mL). NaH (167 mg, 60% active ingredient) was added, and the reaction was stirred at 25°C for 30 min. 13-Bromo-2,5,8,11-tetraoxatridecane (1.13 g) was then added. The reaction mixture was stirred at 25°C for 3 h under nitrogen. The mixture was then poured into 30 mL of water and extracted with ethyl acetate (20 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, concentrated under reduced pressure, and separated by column chromatography (THF / PE, 0–100%) to obtain the title compound (300 mg).

[0376] LC-MS: m / z(ESI):800.6[M+H] + .

[0377] Step 5: Synthesis of tert-butyl 6-(2-(cyclopentanesulfonyl)-5-oxo-8,8-di(2,5,8,11-tetraoxatridecan-13-yl)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)hexanoate (Compound 18)

[0378] Intermediate 18-4 (450 mg) was dissolved in anhydrous dichloromethane (5 mL) at 0°C, and m-chloroperbenzoic acid (388.25 mg, 85% purity) was added. The reaction mixture was stirred at 25°C for 1 h. The mixture was then separated by column chromatography (EA / PE, 0–100%) to obtain the title compound (300 mg).

[0379] LC-MS: m / z(ESI):832.3[M+H] + .

[0380] 1H NMR (400MHz, DMSO-d6) δ = 9.26 (s, 1H), 4.31-4.28 (m, 1H), 3.70 (s, 2H), 3.53-3.39 (m, 24H), 3.38-3.34 (m, 4H), 3.32–3.30 (m, 1H) ),3.23(s,6H),2.20(t,J=7.3Hz,2H),2.13-1.93(m,8H),1.73-1.63(m,4H),1.59–1.55(m,4H),1.39(s,9H),1.33–1.29(m,2H)

[0381] Example 19, tert-Butyl 3-(2'-(cyclopentanesulfonyl)-5'-oxo-5'H-spiro[cyclopenta-1,8'-pyrido[4,3-d]pyrimidin]-6'(7'H)-yl)propanoate (Compound 19)

[0382] Step 1: Synthesis of tert-butyl 3-(2'-(cyclopentylthio)-5'-oxo-5'H-spiro[cyclopentane-1,8'-pyrido[4,3-d]pyrimidin]-6'(7'H)-yl)propanoate (Intermediate 19-1)

[0383] Under a nitrogen atmosphere at 0°C, intermediate 7-1 (1 g) was dissolved in N,N-dimethylformamide (10 mL). NaH (635.7 mg, 60% purity) was added, and the reaction was stirred at 0°C for 15 min. 1,4-diiodobutane (2.46 g) was then added. The reaction mixture was stirred at 0°C under nitrogen for 1 h. The mixture was then poured into 20 mL of ice water and extracted with ethyl acetate (10 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, concentrated under reduced pressure, and separated by column chromatography (ethyl acetate / petroleum ether, 0-25%) to obtain the title compound (620 mg).

[0384] LC-MS: m / z(ESI):432.1[M+H] +

[0385] Step 2: Synthesis of tert-butyl 3-(2'-(cyclopentanesulfonyl)-5'-oxo-5'H-spiro[cyclopentane-1,8'-pyrido[4,3-d]pyrimidin]-6'(7'H)-yl)propanoate (Compound 19)

[0386] Intermediate 19-1 (100 mg) was dissolved in anhydrous dichloromethane (2 mL) at 0°C, and m-chloroperbenzoic acid (188.16 mg, 85% purity) was added. The reaction mixture was stirred at 0°C for 1 h. The mixture was concentrated under reduced pressure and separated by column chromatography (ethyl acetate / petroleum ether, 0-50%) to obtain the title compound (49.2 mg).

[0387] LC-MS: m / z(ESI):464.3[M+H] + .

[0388] 1 H NMR (400MHz, Chloroform-d) δ = 9.35 (s, 1H), 4.34-4.13 (m, 1H), 3.80 (t, J = 6.4Hz, 2H), 3.61 (s, 2H), 2.66 (t, J = 6 .3Hz,2H),2.24-2.11(m,4H),2.05(m,2H),1.99-1.90(m,2H),1.89-1.75(m,6H),1.73-1.65(m,2H),1.45(s,9H)

[0389] Example 20, tert-butyl 3-(2'-(cyclopentanesulfonyl)-5'-oxo-2,3,5,6-tetrahydro-5'H-spiro[pyrano-4,8'-pyrido[4,3-d]pyrimidin]-6'(7'H)-yl)propanoate (Compound 20)

[0390] Step 1: Synthesis of tert-butyl 3-(2'-(cyclopentylthio)-5'-oxo-2,3,5,6-tetrahydro-5'H-spiro[pyrano-4,8'-pyrido[4,3-d]pyrimidin]-6'(7'H)-yl)propanoate (Intermediate 20-1)

[0391] Under a nitrogen atmosphere at 0°C, intermediate 7-1 (200 mg) was dissolved in N,N-dimethylformamide (2 mL). NaH (127.15 mg, 60% active ingredient) and 1-iodo-2-(2-iodoethoxy)ethane (518.01 mg) were added. The reaction mixture was stirred at 0°C under nitrogen for 1 h. Ice water (2 mL) was then added to the reaction mixture, and the mixture was extracted with ethyl acetate (1 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, concentrated under reduced pressure, and separated by column chromatography (ethyl acetate / petroleum ether, 0-60%) to obtain the title compound (110 mg).

[0392] MS m / z(ESI):448.2[M+H] + .

[0393] Step 2: Synthesis of tert-butyl 3-(2'-(cyclopentanesulfonyl)-5'-oxo-2,3,5,6-tetrahydro-5'H-spiro[pyrano-4,8'-pyrido[4,3-d]pyrimidin]-6'(7'H)-yl)propanoate (Compound 20)

[0394] Intermediate 20-1 (60 mg) was dissolved in anhydrous dichloromethane (1 mL) at 0°C, and m-chloroperbenzoic acid (108.86 mg, 85% purity) was added. The reaction mixture was stirred at 25°C for 1 hour. After the reaction was completed, the mixture was concentrated under reduced pressure and separated by column chromatography (ethyl acetate / petroleum ether, 0-55%) to obtain the title compound (9.8 mg).

[0395] MS m / z(ESI):480.3[M+H] + .

[0396] 1 H NMR (400MHz, Chloroform-d) δ = 9.40 (s, 1H), 4.35-4.18 (m, 1H), 4.00 (m, 2H), 3.86-3.72 (m, 6H), 2.68 (t, J= 6.3Hz,2H),2.31-2.23(m,2H),2.18(m,2H),2.06(m,2H),1.93-1.81(m,2H),1.77-1.65(m,4H),1.45(s,9H)

[0397] Example 21, tert-Butyl 3-(2-methanesulfonyl-7-oxo-5,7-dihydro-6H-pyrrolo[3,4-d]pyrimidin-6-yl)propanoate (Compound 21)

[0398] Step 1: Synthesis of methyl 5-(((tert-Butyloxycarbonyl)amino)methyl)-2-methylthiopyrimidine-4-carboxylate (Intermediate 21-1)

[0399] To a mixture of methyl 5-bromo-2-methylthiopyrimidine-4-carboxylate (4 g), potassium [(tert-butoxycarbonylamino)methyl]trifluoroborate (5.41 g), cesium carbonate (9.91 g), palladium acetate (341.34 mg), and n-butyldi(1-adamantyl)phosphine (817.62 mg) was added 1,4-dioxane (50 mL) and water (5 mL) at room temperature under a nitrogen atmosphere. The resulting mixture was stirred at 80°C for 5 h. The solvent was then removed under reduced pressure, and the title compound (1300 mg) was obtained by column chromatography (tetrahydrofuran / petroleum ether, 0-30%).

[0400] LC-MS: m / z(ESI):314.0[M+H] + .

[0401] Step 2: Synthesis of (4-methoxycarbonyl-2-methylthiopyrimidin-4-yl)methylamine hydrochloride (Intermediate 21-2)

[0402] To a solution of Intermediate 21-1 (1300 mg) in 20 mL of ethyl acetate was added 4 M hydrogen chloride in ethyl acetate (20 mL) at room temperature under nitrogen. The resulting mixture was stirred at room temperature for 4 h. After completion of the reaction as determined by LCMS, the mixture was filtered and the filter cake was washed with ethyl acetate to afford the title compound (750 mg).

[0403] LC-MS: m / z(ESI):214.0[M+H] + .

[0404] Step 3: Synthesis of tert-butyl 3-(2-methylthio-7-oxo-5,7-dihydro-6H-pyrrolo[3,4-d]pyrimidin-6-yl)propanoate (Intermediate 21-3)

[0405] Under a nitrogen atmosphere, a mixture of intermediate 21-2 (100 mg), cesium carbonate (195.71 mg), tert-butyl 3-bromopropionate (83.73 mg), and N,N-dimethylformamide (4 mL) was heated to 80°C for 16 h. The solvent was then removed under reduced pressure, and the title compound (20 mg) was obtained by column chromatography (tetrahydrofuran / petroleum ether, 0-50%).

[0406] LC-MS: m / z(ESI):310.0[M+H] +

[0407] Step 4: Synthesis of tert-butyl 3-(2-methanesulfonyl-7-oxo-5,7-dihydro-6H-pyrrolo[3,4-d]pyrimidin-6-yl)propanoate (Compound 21)

[0408] At 0°C, m-chloroperbenzoic acid (33.47 mg) was added to a solution of intermediate 21-3 (20 mg) in dichloromethane (4 mL). The resulting mixture was stirred at room temperature for 2 h. The reaction solution was directly purified by HPLC (Phenomenex Gemini NX 150×30 mm, 5 μm column; mobile phase: [A: water (ammonium bicarbonate), B: acetonitrile]; B%: 13%-43%, 11 min) to obtain the title compound (2.9 mg).

[0409] (Mobile phase A preparation: 30L pure water + 24g ammonium bicarbonate)

[0410] 1 H NMR (400MHz, DMSO-d6) δ = 9.44 (s, 1H), 4.77-4.66 (m, 2H), 3.90-3.78 (m, 2H), 3.50-3.45 (m, 3H), 2.69-2.63 (m, 2H), 1.41-1.38 (m, 9H).

[0411] Preparation Examples of Drug-Linkers (Examples 22-40)

[0412] Example 22, N-((4S,12S)-12-benzyl-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl-2,2-d2)-3,8,11,14,17-pentaoxo-5-oxa-2,7,10,13,16-pentaazaoctadec-18-yl)-6-(2-methylsulfonyl-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)hexanamide (a-L1-1)

[0413] Step 1: Synthesis of 1-(Benzo[d][1,3]dioxol-5-yl-2,2-d2)ethan-1-one (Intermediate L1-1)

[0414] 3',4'-Dihydroxyacetophenone (3g) was dissolved in anhydrous DMF (25mL), and deuterated dichloromethane (8.57g) and potassium carbonate (8.18g) were added. After addition, the temperature was raised to 90°C and stirred for 16h. The reaction solution was then added to water (100mL) and extracted with ethyl acetate (200mL*2). The combined organic phases were washed with saturated brine (100mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated to dryness under reduced pressure, and the residue was purified by column chromatography (ethyl acetate / petroleum ether = 5:1) to obtain the title compound (2.4g).

[0415] MS m / z(ESI):167.1[M+H] + .

[0416] Step 2: Synthesis of 1-(6-nitrobenzo[d][1,3]dioxol-5-yl-2,2-d2)ethan-1-one (Intermediate L1-2)

[0417] Intermediate L1-1 (2.4 g) was dissolved in anhydrous acetic acid (10 mL). Concentrated nitric acid (32.50 g, 70% content) was added dropwise at 0°C, followed by stirring at 0°C for 10 minutes. The mixture was then warmed to room temperature and stirred for 1 hour. After completion of the reaction, the reaction mixture was added dropwise to ice water (200 mL), filtered, and the filter cake dried to yield the title compound (1.9 g).

[0418] MS m / z(ESI):212.0[M+H] + .

[0419] 1H NMR (400MHz, DMSO-d6) δ7.69(s,1H),7.30(s,1H),2.49(s,3H).

[0420] Step 3: Synthesis of N-(6-acetylbenzo[d][1,3]dioxol-5-yl-2,2-d2)acetamide (Intermediate L1-3)

[0421] Intermediate L1-2 (1.8 g) was dissolved in acetic acid (25 mL), and acetic anhydride (1.84 g) and reduced iron powder (4.76 g) were added. The mixture was stirred at room temperature for 1 h. After completion of the reaction, the mixture was filtered, and the filtrate was concentrated to dryness under reduced pressure. The residue was purified by column chromatography (ethyl acetate / petroleum ether = 5:1) to obtain the title compound (1.5 g).

[0422] MS m / z(ESI):224.1[M+H] + .

[0423] Step 4: Synthesis of N-(6-(2-bromoacetyl)benzo[d][1,3]dioxol-5-yl-2,2-d2)acetamide (Intermediate L1-4)

[0424] A solution of HBr in acetic acid (2.39 g, 33% content) was added dropwise to a solution of intermediate L1-3 (1.45 g) in anhydrous acetic acid (25 mL), and then Br was added dropwise. 2( After the reaction was complete, the reaction solution was concentrated to dryness under reduced pressure, and the residue was added to water (50 mL) and extracted with ethyl acetate (50 mL*2). The organic phase was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and filtered. The residue was purified by column chromatography (ethyl acetate / petroleum ether = 5:1) to obtain the title compound (1.3 g).

[0425] MS m / z(ESI):302.1[M+H] + .

[0426] Step 5: Synthesis of 1-(6-aminobenzo[d][1,3]dioxol-5-yl-2,2-d2)-2-chloroethane-1-one (Intermediate L1-5)

[0427] Intermediate L1-4 (1.2 g) and concentrated hydrochloric acid (144.82 mg) were dissolved in ethanol (15 mL), and the reaction mixture was stirred at 60° C. for 16 h. After completion of the reaction, the reaction mixture was concentrated to dryness under reduced pressure, and the residue was purified by high-performance liquid chromatography (YMC-Actus Triart C18 column, 5 μm silica, 30 mm diameter, 150 mm length; using decreasingly polar mixtures of water (containing 0.05% NH 4 HCO 3 ) and acetonitrile as eluents (acetonitrile gradient ratio 40%-50%) to obtain the title compound (577 mg).

[0428] MS m / z(ESI):216.0[M+H] + .

[0429] Step 6: Synthesis of (S)-14-(chloromethyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione-2,2-d2 (Intermediate L1-6)

[0430] Intermediate L1-5 (100.0 mg) and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (109.87 mg) were dissolved in toluene (1 mL) and acetic acid (1 mL). Pyridinium p-toluenesulfonate (5.24 mg) was added. The reaction mixture was stirred at 100°C for 16 h. After the reaction was completed, the reaction mixture was cooled to room temperature and concentrated to dryness under reduced pressure. Ethanol (5 mL) was added, and the reaction mixture was stirred at 25°C for 0.5 h. The reaction mixture was filtered, and the filter cake was washed with ethanol (5 mL x 2) to obtain the title compound (100.0 mg).

[0431] MS m / z(ESI):443.0[M+H] + .

[0432] Step 7: Synthesis of (S)-14-(aminomethyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione-2,2-d2 (Intermediate L1-7)

[0433] Intermediate L1-6 (100.00 mg) was dissolved in anhydrous ethanol (1.5 mL) and anhydrous DMF (1.5 mL), and hexamethylenetetramine (94.97 mg) was added. The reaction mixture was stirred at 50°C for 6 h. After completion of the reaction, the reaction mixture was concentrated to dryness under reduced pressure, and the residue was purified by high-performance liquid chromatography (column: Boston Green ODS 150*30 mm*5 μm; mobile phase: [A: water (formic acid), B: acetonitrile]; B%: 0%-30%, 12 min) to obtain the title compound (25.0 mg).

[0434] MS m / z(ESI):424.0[M+H] + .

[0435] Step 8: Synthesis of (S)-4-benzyl-3-(2-cyclopropylacetyl)oxazolidin-2-one (Intermediate L1-10)

[0436] Weigh compound L1-8 (150.0 g), DMAP (160.15 g), and compound L1-9 (221.2 g), dissolve in 1500 mL of dichloromethane, and stir at room temperature for 15 min. Weigh EDCI (359.0 g) and add the reaction solution in batches. After the addition is complete, stir at room temperature for about 5 h. After the reaction is completed, dichloromethane (1500 mL) is added to the reaction solution to dilute it, and then washed with water (500 mL) twice, 2N HCl (500 mL) once, saturated sodium bicarbonate solution (500 mL) once, and saturated saline solution (500 mL) once. The organic phase is dried over anhydrous sodium sulfate, filtered, and the filtrate is concentrated to dryness under reduced pressure to obtain the title compound (302 g).

[0437] 1 H NMR (400MHz, CDCl3) δ7.32-7.35(m,2H),7.26-7.29(m,1H),7.21-7.23(m,2H),4.68-4.71(m,1H),4.17-4.23( m,2H),3.30-3.33(dd,1H),2.91-2.95(dd,1H),2.78-2.82(m,2H),1.14-1.18(m,1H),0.59-0.63(m,2H),0.21- 0.26(m,2H).

[0438] Step 9: Synthesis of (S)-4-benzyl-3-((S)-2-cyclopropyl-2-hydroxyacetyl)oxazolidin-2-one (Intermediate L1-12)

[0439] Intermediate L1-10 (200 g) was weighed and dissolved in 2000 mL of anhydrous THF. Under nitrogen, the mixture was stirred at -78°C for 15 min. Subsequently, sodium bis(trimethylsilyl)amide (443.5 mL, 2 M in THF) was added dropwise to the reaction mixture. After the addition was complete, the reaction mixture was stirred at -78°C for 30 min. Intermediate L1-11 (201.5 g) was dissolved in 700 mL of THF until clear and slowly added dropwise to the reaction mixture. After the addition was complete, the mixture was stirred at -78°C for 2 h. Subsequently, 220 mL of glacial acetic acid was added to the reaction mixture to quench the reaction. After the addition was complete, the mixture was gradually warmed to room temperature and 600 mL of 2N HCl was added to the reaction mixture. The mixture was stirred at room temperature (20-25°C) for 10 h. The reaction mixture was then concentrated under reduced pressure, and ethyl acetate (1000 mL) and water (200 mL) were added to the residue and stirred for 20 min. The aqueous phase was separated and extracted twice with ethyl acetate (500 mL x 2). The organic phases were combined and washed twice with saturated NaHCO3 solution 400 mL, saturated Na2S2O3 solution 400 mL and saturated brine, respectively. The resulting organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 1 / 30) to obtain the title compound (128.7 g).

[0440] 1 H NMR (400MHz, CDCl3) δ7.37 (dd, J=8.1, 6.8Hz, 2H), 7.33-7.29 (m, 1H), 7.27-7.23 (m ,2H),4.81(dd,J=7.9,5.9Hz,1H),4.72(ddt,J=10.0,7.5,2.9Hz,1H),4.33(t,J=8 .3Hz,1H),4.28(dd,J=9.1,2.5Hz,1H),3.48(dd,J=8.2,3.9Hz,1H),3.35(dd,J=13 .5,3.4Hz,1H),2.88(dd,J=13.5,9.4Hz,1H),1.36-1.29(m,1H),0.62-0.44(m,4H).

[0441] Step 10: Synthesis of (S)-4-benzyl-3-((S)-2-((tert-butyldimethylsilyl)oxy)-2-cyclopropylacetyl)oxazolidin-2-one (Intermediate L1-13)

[0442] Intermediate L1-12 (128.7 g) was weighed and dissolved in 1300 mL of DCM. Imidazole (56.17 g) was added and stirred in an ice bath for 15 min. TBSCl (107.3 g) was then added portionwise to the reaction mixture and stirred at room temperature for 3 h. 200 mL of 2N HCl was added to the reaction mixture and stirred for 20 min. The mixture was then separated. The organic phase was washed twice with 200 mL of saturated NaHCO₃ solution and twice with saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 80:1) to obtain the title compound (160 g).

[0443] 1 H NMR (400MHz, CDCl3) δ7.35-7.37(m,2H),7.30-7.32(m,1H),7.26-7.29(m,2H),5.26-5.31(m,1H),4.67-4.71(m,1H),4.20-4.26(m ,2H),3.41-3.44(dd,1H),2.72-2.76(dd,1H),1.25-1.31(m,1H),0.94(s,9H),0.54-0.56(m,2H),0.46-0.48(m,2H),0.12(s,6H).

[0444] Step 11: Synthesis of (S)-2-((tert-butyldimethylsilyl)oxy)-2-cyclopropylbenzyl acetate (Intermediate L1-14)

[0445] Weigh benzyl alcohol (62.18 g) and dissolve it in 500 mL of THF. Stir at -25°C. Weigh n-butyl lithium (213.6 mL, 2.5 M in THF) and slowly add it dropwise to the reaction mixture. Stir at -25°C for 1 hour. Weigh intermediate L1-13 (160 g) and dissolve it in 320 mL of THF. Slowly add it dropwise to the reaction mixture at -25°C. Stir at -15°C for 3 hours. Add saturated NH4Cl (200 mL) to the reaction mixture to quench the reaction. The mixture was then concentrated under reduced pressure, and 400 mL of methyl tert-butyl ether and water (150 mL) were added to the reaction solution, stirred for 30 min, and separated. The aqueous phase was extracted twice with methyl tert-butyl ether (200 mL x 2), and the organic phases were combined, washed once with saturated brine (250 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 100: 1) to obtain the title compound (125 g).

[0446] 1H NMR (400MHz, CDCl3) δ7.28-7.40(m,5H),5.17-5.26(m,2H),3.86-3.89(m,1H),1.21-1.46(m,1H),0.90(s,9H),0.45-0.51(m,4H),0.05(s,6H).

[0447] Step 12: Synthesis of (S)-2-cyclopropyl-2-hydroxybenzyl acetate (Intermediate L1-15)

[0448] Intermediate L1-14 (125 g) was weighed and dissolved in 1200 mL of THF. Glacial acetic acid (35.1 g) was added and stirred at room temperature for 5 min. TBAF (585 mL, 1 M in THF) was then added to the reaction solution, and the reaction solution was placed at 45°C for 4 h. The reaction solution was concentrated under reduced pressure to remove the solvent, and 300 mL of water and 400 mL of methyl tert-butyl ether were added to the residue. The mixture was stirred for 20 min and separated. The aqueous phase was extracted twice with methyl tert-butyl ether (200 mL). The organic phases were combined and washed twice with 200 mL of saturated NaHCO3 solution and saturated brine. The organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 60:1) to obtain the title compound (68.9 g).

[0449] 1 H NMR (400MHz, CDCl3) δ7.27-7.39(m,5H),5.22-5.28(m,2H),3.82(d,1H),2.7(brs,1H),1.11-1.15(m,1H),0.41-0.56(m,4H).

[0450] Intermediate L1-15 was further analyzed by the following chiral supercritical fluid chromatography conditions.

[0451] Under the above chiral supercritical fluid chromatography conditions, the retention time of intermediate L1-15 is 3.013 minutes.

[0452] Step 13: Synthesis of (2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)methyl acetate (Intermediate L1-17)

[0453] Compound L1-16 (25 g), lead acetate (43.79 g) and pyridine (6.98 g) were dissolved in a mixed solvent of tetrahydrofuran (600 mL) and toluene (200 mL). The mixture was replaced with nitrogen three times. The reaction was heated to 85°C under nitrogen protection and stirred for 18 hours. The mixture was then cooled to room temperature, filtered, and the filtrate was concentrated to dryness under reduced pressure. The crude product was purified by rapid silica gel column chromatography ( 330g Silica Flash Column, mobile phase gradient 0-75% ethyl acetate / petroleum ether, flow rate 100 mL / min) to obtain the title compound (18 g).

[0454] ((MS m / z(ESI):391.1[M+Na] + .

[0455] Step 14: Synthesis of (S)-10-cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-carboxylic acid benzyl ester (Intermediate L1-18)

[0456] Intermediate L1-17 (5 g), intermediate L1-15 (8.40 g), and p-toluenesulfonic acid pyridinium salt (341.09 mg) were dissolved in dichloromethane (150 mL), and the atmosphere was replaced with nitrogen three times. The reaction solution was heated to 65 ° C under nitrogen protection and stirred for 48 hours. Then it was cooled to room temperature, the reaction solution was filtered, and the filtrate was concentrated to dryness under reduced pressure. The crude product was purified by rapid silica gel column ( 120g Silica Flash Column, mobile phase gradient 0-45% ethyl acetate / petroleum ether, flow rate 80 mL / min), and further purified by HPLC (column: Boston Prime C18 150*30mm*5um; mobile phase: [A: water (formic acid), B: acetonitrile]; B%: 42%-82%, 13 min) to obtain the title compound (1.4 g).

[0457] MS m / z(ESI):537.2[M+Na] + .

[0458] Step 15: Synthesis of (S)-10-cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-carboxylic acid (Intermediate L1-19)

[0459] Intermediate L1-18 (1.4 g) was dissolved in a mixed solvent of methanol (15 mL) and water (15 mL). Wet palladium on carbon (10% by mass, 0.15 g) was added under a nitrogen atmosphere. The atmosphere was replaced with hydrogen three times, and the reaction mixture was stirred at 25°C under a hydrogen atmosphere (15 psi) for 16 hours. After completion of the reaction, the reaction mixture was filtered, and the filtrate was concentrated to dryness under reduced pressure. The crude product was purified by HPLC (column: Boston Prime C18 150*30mm*5μm; mobile phase: [A: water (formic acid), B: acetonitrile]; B%: 24%-64%, 13 min) to obtain the title compound.

[0460] LCMS m / z(ESI):447.5[M+Na] + .

[0461] Step 16: Synthesis of resin-supported (S)-10-cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-carboxylic acid (Intermediate L1-20)

[0462] CTC resin (specification: approximately 1.19 mmol / g) (257 mg) was added to dichloromethane (3 mL), followed by intermediate L1-19 (130 mg) and diisopropylethylamine (59.37 mg). The reaction mixture was nitrogen-purged three times and shaken on a shaker at 25°C for 16 h. After completion of the reaction, the resin was washed three times with methanol (10 mL) and then dichloromethane (10 mL). The resin was then filtered to dryness and dried under vacuum to yield the title compound (330 mg).

[0463] Step 17: Synthesis of resin-supported (S)-2-((2-aminoacetamido)methoxy)-2-cyclopropylacetic acid (Intermediate L1-21)

[0464] Intermediate L1-20 (330 mg) was dissolved in N,N-dimethylformamide (5 mL) and piperidine (1.08 g) was added. The reaction mixture was shaken at 25°C for 1 hour. After the reaction, the resin was washed with methanol (10 mL) and dichloromethane (10 mL) three times, followed by filtration to dryness and drying under vacuum to obtain the title compound (220 mg).

[0465] Step 18: Synthesis of resin-supported (5S)-5-benzyl-13-cyclopropyl-1-(9H-fluoren-9-yl)-3,6,9-trioxy-2,12-dioxa-4,7,10-triazatetradecane-14-oic acid (Intermediate L1-22)

[0466] Intermediate L1-21 (220 mg) and (((9H-fluoren-9-yl)methoxy)carbonyl)-L-phenylalanine (230.17 mg) were dissolved in N,N-dimethylformamide (5 mL). O-benzotriazole-tetramethyluronium hexafluorophosphate (225.31 mg) and diisopropylethylamine (99.96 mg) were added. The reaction mixture was shaken at 25°C for 1 hour. After completion of the reaction, the resin was washed with methanol (10 mL) and dichloromethane (10 mL) three times, followed by filtration to dryness and drying under vacuum to obtain the title compound (377 mg).

[0467] Step 19: Synthesis of resin-supported (S)-2-((2-((S)-2-amino-3-phenylpropionamido))acetamido)methoxy)-2-cyclopropylacetic acid (Intermediate L1-23)

[0468] Intermediate L1-22 (377 mg) was dissolved in N,N-dimethylformamide (5 mL) and piperidine (37.22 mg, 437.15 μmol, 43.17 μL) was added. The reaction mixture was shaken at 25°C for 1 hour. After completion of the reaction, the resin was washed with methanol (10 mL) and dichloromethane (10 mL) three times, followed by filtration to dryness and drying under vacuum to obtain the title compound (270 mg).

[0469] Step 20: Synthesis of resin-supported (11S,19S)-11-benzyl-19-cyclopropyl-1-(9H-fluoren-9-yl)-3,6,9,12,15-pentaoxo-2,18-dioxa-4,7,10,13,16-pentaazaeicosane-20-carboxylic acid (Intermediate L1-24)

[0470] Intermediate L1-23 (270 mg) was dissolved in N,N-dimethylformamide (5 mL), followed by intermediate L1-16 (209.58 mg), O-benzotriazole-tetramethyluronium hexafluorophosphate (224.29 mg), and diisopropylethylamine (99.51 mg). The reaction mixture was shaken at 25°C for 1 hour. After the reaction, the resin was washed with methanol (10 mL) and dichloromethane (10 mL) three times, followed by filtration to dryness and drying under vacuum to obtain the title compound (403 mg).

[0471] Step 21: Synthesis of resin-supported (10S)-16-amino-10-benzyl-2-cyclopropyl-6,9,12,15-tetraoxy-3-oxy-5,8,11,14-tetraazahexadecane-1-carboxylic acid (Intermediate L1-25)

[0472] Intermediate L1-24 (403 mg) was dissolved in N,N-dimethylformamide (5 mL) and piperidine (1.08 g) was added. The reaction mixture was shaken at 25°C for 1 hour. After the reaction, the resin was washed with methanol (10 mL) and dichloromethane (10 mL) three times, followed by filtration to dryness and drying under vacuum to obtain the title compound (300 mg).

[0473] Step 22: Synthesis of 6-(2-methylsulfonyl-5-oxo-5H,6H,7H,8H-pyrido[4,3-d]pyrimidin-6-yl)hexanoic acid (Intermediate L1-26)

[0474] Intermediate 18-2 (2.4 g) was dissolved in dichloromethane (50 mL) and trifluoroacetic acid (10 mL) was added. The reaction mixture was stirred at 25°C for 1 h. The solvent was then removed by concentration under reduced pressure. Water (20 mL) was added, stirred at room temperature for 5 minutes, and filtered to obtain the title compound (1.5 g).

[0475] (MS m / z(ESI):342.1[M+H] + .

[0476] Step 23: Synthesis of resin-supported (2S,10S)-10-benzyl-2-cyclopropyl-23-(2-methylsulfonyl-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)-6,9,12,15,18-pentaoxo-3-oxa-5,8,11,14,17-pentaazatricosanoic acid (Intermediate L1-27)

[0477] Intermediate L1-25 (200 mg) was added to DMF (3 mL), followed by Intermediate L1-26 (127 mg), O-benzotriazole-tetramethyluronium hexafluorophosphate (141 mg), and diisopropylethylamine (69 mg). The reaction mixture was shaken at 25°C for 1 hour. After completion of the reaction, the resin was washed with methanol (10 mL) and dichloromethane (10 mL) three times, followed by filtration to dryness and drying under vacuum to obtain the title compound (220 mg).

[0478] Step 24: Synthesis of (2S,10S)-10-benzyl-2-cyclopropyl-23-(2-methylsulfonyl-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)-6,9,12,15,18-pentaoxo-3-oxa-5,8,11,14,17-pentaazatricosanoic acid (Intermediate L1-28)

[0479] Intermediate L1-27 (220 mg) was added to a mixture of dichloromethane (4 mL) and 1,1,1,3,3,3-hexafluoroisopropanol (1 mL) and shaken at 25°C for 1 h. After the reaction, the reaction mixture was filtered to remove the resin, and the filtrate was concentrated to dryness under reduced pressure to obtain the title compound (17 mg).

[0480] Step 25: Synthesis of N-((4S,12S)-12-benzyl-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl-2,2-d2)-3,8,11,14,17-pentaoxo-5-oxa-2,7,10,13,16-pentaazaoctadec-18-yl)-6-(2-methylsulfonyl-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)hexanamide (a-L1-1)

[0481] Intermediate L1-28 (17 mg), intermediate L1-7 (10.06 mg), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (8.28 mg), pyridine (5.13 mg), and 1-hydroxybenzotriazole (5.84 mg) were dissolved in DMF (0.7 mL). The atmosphere was purged with nitrogen three times. The reaction mixture was stirred at 25°C under nitrogen for 1 hour. After completion of the reaction, the reaction mixture was directly purified by HPLC (column: Welch Xtimate C18 150*25mm*5um; mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 25%-55%, 11 min) to obtain the title compound (2.1 mg).

[0482] 1H NMR (400MHz, DMSO-d6) δ = 9.22 (s, 1H), 8.67 (t, J = 6.0Hz, 1H), 8.60 (t, J = 6. 5Hz,1H),8.30(t,J=5.7Hz,1H),8.17-8.05(m,2H),8.05-7.97(m,1H),7.80 (s,1H),7.52(s,1H),7.29-7.20(m,5H),7.18-7.16(m,1H),6.54-6.46(m, 1H),5.44(d,J=8.7Hz,4H),4.85-4.71(m,2H),4.68-4.66(m,1H),4.55-4.4 5(m,1H),4.42-4.39(m,1H),3.77-3.63(m,7H),3.62-3.53(m,1H),3.51-3 .45(m,3H),3.43(s,3H),3.25(t,J=6.7Hz,2H),3.05-3.03(m,1H),2.80-2. 78(m,1H),2.13(t,J=7.5Hz,2H),1.94-1.79(m,2H),1.61-1.46(m,4H),1.3 3-1.21(m,2H),1.01-0.92(m,1H),0.87(t,J=7.3Hz,3H),0.43-0.26(m,4H)

[0483] MS m / z(ESI):1192.6[M+H] + .

[0484] Example 23, N-((4S,12S,21S)-12-benzyl-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolane-[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl-2,2-d2)-21-(3-(2 -(methylsulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propionylamino)-3,8,11,14,17,20-hexaoxo-5-oxa-2,7,10,13,16,19-hexaazapentacosan-25-yl)-2,5,8,11,14,17,20,23-octaoxahexacosan-26-amide (a-L2-1)

[0485] Step 1: Synthesis of (S)-32-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azatricarboxane-33-oic acid (Intermediate L2-1)

[0486] 2,5-Dioxopyrrolidin-1-yl 2,5,8,11,14,17,20,23-octaoxacohexacosa-26-anoate (4.5 g) was dissolved in DMF (70 mL) and (((9H-fluoren-9-yl)methoxy)carbonyl)-L-lysine (3.25 g) was added. The reaction solution was stirred at 25 ° C for 16 hours. After the reaction was completed, water (300 mL) was added to the reaction solution, and ethyl acetate was used for extraction (50 mL * 3 times). The organic phases were combined and dried over anhydrous sodium sulfate. After filtration, the organic phase was concentrated under reduced pressure to remove the solvent, and the residue was purified by rapid silica gel column ( 40g Silica Flash column chromatography, gradient 0-4% methanol / dichloromethane @ 60 mL / min) to give the title compound (4.1 g)

[0487] MS m / z(ESI):763.3[M+H] + .

[0488] Step 2: Synthesis of resin-supported (32S,41S,49S)-32-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-41-benzyl-49-cyclopropyl-26,33,36,39,42,45-hexaoxo-2,5,8,11,14,17,20,23,48-nonaoxa-27,34,37,40,43,46-hexaazapentacontan-50-oic acid (Intermediate L2-2)

[0489] Intermediate L1-25 (1.5 g) was dissolved in DMF (15 mL), followed by Intermediate L2-1 (2.02 g), O-benzotriazole-tetramethyluronium hexafluorophosphate (1.01 g), and diisopropylethylamine (771.05 mg). The reaction mixture was shaken at 25°C for 1 hour. After the reaction, the resin was washed with methanol (20 mL) and dichloromethane (20 mL) three times, followed by filtration to dryness and drying under vacuum to obtain the title compound (1.67 g).

[0490] MS m / z(ESI):1230.5[M+Na] +

[0491] Step 3: Synthesis of resin-supported (32S,41S,49S)-32-amino-41-benzyl-49-cyclopropyl-26,33,36,39,42,45-hexaoxo-2,5,8,11,14,17,20,23,48-nonaoxa-27,34,37,40,43,46-hexaazapentacontan-50-oic acid (Intermediate L2-3)

[0492] Intermediate L2-2 (1.67 g) was dissolved in DMF (10 mL) and piperidine (2.5 mL) was added. The reaction mixture was shaken at 25°C for 1 hour. After the reaction, the resin was washed with methanol (20 mL) and dichloromethane (20 mL) three times, followed by filtration to dryness and drying under vacuum to obtain the title compound (1.5 g).

[0493] MS m / z(ESI):986.5[M+H] + .

[0494] Step 4: Synthesis of 3-(2-(methylsulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)-propionic acid (Intermediate L2-4)

[0495] Under a nitrogen atmosphere, compound 1 (400 mg) was dissolved in 20 mL of anhydrous dichloromethane and cooled to -78°C. At this temperature, 5 mL of trifluoroacetic acid was added, and the mixture was slowly warmed to room temperature. The solvent was then removed under a stream of nitrogen. 5 mL of anhydrous acetonitrile was added to the residue, which was filtered. The filter cake was washed with a 1:1 mixture of petroleum ether and ethyl acetate to obtain the title compound (220 mg).

[0496] Step 5: Synthesis of resin-supported (32S,41S,49S)-41-benzyl-49-cyclopropyl-32-(3-(2-(methylsulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propionamido)-26,33,36,39,42,45-hexaoxo-2,5,8,11,14,17,20,23,48-nonaoxa-27,34,37,40,43,46-hexaazapentacontan-50-oic acid (Intermediate L2-5)

[0497] Intermediate L2-3 (300 mg) was dissolved in DMF (5 mL), and intermediate L2-4 (93.52 mg), O-benzotriazole-tetramethyluronium hexafluorophosphate (119.29 mg), and diisopropylethylamine (91.09 mg) were added sequentially. The reaction solution was shaken at 25°C for 1 hour. After completion of the reaction, the resin was washed with methanol (10 mL) and dichloromethane (10 mL) three times, followed by filtration to dryness and drying under vacuum to obtain the title compound (325 mg).

[0498] Step 6: Synthesis of (32S,41S,49S)-41-benzyl-49-cyclopropyl-32-(3-(2-(methylsulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propionamido)-26,33,36,39,42,45-hexaoxo-2,5,8,11,14,17,20,23,48-nonaoxa-27,34,37,40,43,46-hexaazapentacontan-50-oic acid (Intermediate L2-6)

[0499] Intermediate L2-5 (325 mg) was added to a mixed solvent of dichloromethane (10 mL) and 1,1,1,3,3,3-hexafluoroisopropanol (2.5 mL) and shaken at 25°C for 0.5 h. After the reaction, the reaction mixture was filtered to remove the resin, and the filtrate was concentrated to dryness under reduced pressure to obtain the title compound (75 mg).

[0500] MS m / z(ESI):562.0[(M+H + ) / 2] + .

[0501] Step 7: N-((4S,12S,21S)-12-benzyl-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl-2,2-d2)-21-(3-(2-(methyl)-1-yl)-4-cyclopropyl)-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl-2,2-d2)- Synthesis of (a-L2-1)-3,8,11,14,17,20-hexaoxo-5-oxa-2,7,10,13,16,19-hexaazapentacosan-25-yl)-2,5,8,11,14,17,20,23-octaoxahexacosan-26-amide (a-L2-1)

[0502] Intermediate L2-6 (70 mg), intermediate L1-7 (28.06 mg), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (21.18 mg), 1-hydroxybenzotriazole (14.93 mg), and pyridine (13.11 mg) were dissolved in DMF (0.5 mL). The atmosphere was purged with nitrogen three times. The reaction mixture was stirred at 25°C under nitrogen for 1 hour. After completion of the reaction, the reaction mixture was directly purified by HPLC (column: C18 100×10 mm; mobile phase: [A: water (trifluoroacetic acid), B: acetonitrile]; B%: 16%-46%, 8 min) to obtain the title compound (4.4 mg).

[0503] 1 H NMR (400MHz, DMSO-d6) δ = 9.22 (s, 1H), 8.67 (t, J = 5.9Hz, 1H), 8.60 (t, J = 6.3Hz, 1H), 8.3 2(s,1H),8.26-8.16(m,2H),8.11(d,J=7.9Hz,1H),7.97(t,J=5.4Hz,1H),7.83-7.74(m, 2H),7.54-7.49(m,1H),7.28-7.20(m,5H),7.19-7.14(m,1H),7.12(s,1H),6.50(s,1H), 5.43(d,J=5.3Hz,4H),4.84-4.62(m,3H),4.55-4.47(m,1H),4.42-4.39(m,1H),4.26-4. 13(m,1H),3.78-3.61(m,10H),3.57(t,J=6.6Hz,3H),3.53-3.45(m,23H),3.45-3.40(m, 6H),3.24(s,4H),3.14-3.01(m,3H),2.96(d,J=5.8Hz,2H),2.84-2.74(m,1H),2.28(t,J =6.4Hz,2H),1.95-1.80(m,2H),1.62(s,1H),1.48(d,J=9.4Hz,1H),1.33(s,2H),1.24(s ,1H),1.18(t,J=7.3Hz,3H),1.02-0.93(m,1H),0.88(t,J=7.3Hz,3H),0.41-0.29(m,4H)

[0504] MS m / z(ESI):837.4[(M+2H) / 2] + .

[0505] Example 24, N-((4S,12S,21S)-12-benzyl-21-(3-(2-(cyclopentanesulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propionamido)-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolane-[ 4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl-2,2-d2)-3,8,11,14,17,20-hexaoxo-5-oxa-2,7,10,13,16,19-hexaazapentacosan-25-yl)-2,5,8,11,14,17,20,23-octaoxahexacosan-26-amide (b-L2-1)

[0506] Step 1: Synthesis of 3-(2-cyclopentanesulfonyl-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propanoic acid (Intermediate L3-1)

[0507] To a mixture of compound 7 (135 mg) and DCM (10 mL) was added trifluoroacetic acid (2 mL) at 0°C, and the resulting mixture was allowed to warm to room temperature for 2 h. The solvent was then removed under reduced pressure, and the mixture was separated by column chromatography (EA / PE, 0–100%) to obtain the title compound (80 mg).

[0508] LC-MS: m / z(ESI):354.4[M+H] + .

[0509] Step 2: Synthesis of resin-supported (32S,41S,49S)-41-benzyl-32-(3-(2-(cyclopentanesulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propionamido)-49-cyclopropyl-26,33,36,39,42,45-hexaoxo-2,5,8,11,14,17,20,23,48-nonaoxa-27,34,37,40,43,46-hexaazapentacontan-50-oic acid (Intermediate L3-2)

[0510] Intermediate L2-3 (200 mg) was dissolved in DMF (5 mL), and intermediate L3-1 (73.62 mg), O-benzotriazole-tetramethyluronium hexafluorophosphate (79.53 mg), and diisopropylethylamine (60.73 mg) were added sequentially. The reaction solution was shaken at 25°C for 1 hour. After the reaction, the resin was washed with methanol (10 mL) and dichloromethane (10 mL) three times, followed by filtration to dryness and drying under vacuum to obtain the title compound (285 mg).

[0511] MS m / z(ESI):1344.7[M+Na] + .

[0512] Step 3: Synthesis of (32S,41S,49S)-41-benzyl-32-(3-(2-(cyclopentanesulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propionamido)-49-cyclopropyl-26,33,36,39,42,45-hexaoxo-2,5,8,11,14,17,20,23,48-nonaoxa-27,34,37,40,43,46-hexaazapentacontan-50-oic acid (Intermediate L3-3)

[0513] Intermediate L3-2 (200 mg) was added to a mixed solvent of dichloromethane (10 mL) and 1,1,1,3,3,3-hexafluoroisopropanol (2.5 mL) and shaken at 25°C for 0.5 h. After the reaction, the reaction mixture was filtered to remove the resin, and the filtrate was concentrated to dryness under reduced pressure to obtain the title compound (55 mg).

[0514] MS m / z(ESI):1344.7[M+Na] + .

[0515] Step 4: N-((4S,12S,21S)-12-benzyl-21-(3-(2-(cyclopentanesulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propionamido)-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolane[4, Synthesis of (5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl-2,2-d2)-3,8,11,14,17,20-hexaoxo-5-oxa-2,7,10,13,16,19-hexaazapentacosan-25-yl)-2,5,8,11,14,17,20,23-octaoxahexacosan-26-amide (b-L2-1)

[0516] Intermediate L3-3 (55 mg), intermediate L1-7 (21.15 mg), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (15.96 mg), 1-hydroxybenzotriazole (11.25 mg), and pyridine (9.88 mg) were dissolved in DMF (0.5 mL). The atmosphere was purged with nitrogen three times. The reaction mixture was stirred at 25°C under nitrogen for 1 hour. After completion of the reaction, the reaction mixture was directly purified by HPLC (column: C18 150 × 30 mm × 5 μm; mobile phase: [A: water (trifluoroacetic acid), B: acetonitrile]; B%: 27%-67%, 11 min) to obtain the title compound (6.45 mg).

[0517] 1H NMR (400MHz, DMSO-d6) δ = 9.23 (s, 1H), 8.70-8.64 (m, 1H), 8.63-8.55 (m, 1H), 8.36- 8.28(m,1H),8.26-8.15(m,2H),8.14-8.08(m,1H),8.00-7.94(m,1H),7.82-7.74( m,2H),7.54-7.48(m,1H),7.27-7.21(m,6H),7.19-7.13(m,1H),6.51-6.47(m,1H) ,5.48-5.39(m,4H),4.84-4.72(m,2H),4.71-4.64(m,1H),4.55-4.49(m,1H),4.41- 4.39(m,1H),4.27-4.16(m,2H),3.76-3.63(m,11H),3.57(t,J=6.5Hz,2H),3.51-3.47 (m,26H),3.43(d,J=5.3Hz,3H),3.24(s,3H),3.22-3.17(m,1H),3.07-2.96(m,3H),2. 84-2.76(m,1H),2.31-2.26(m,2H),2.01-1.84(m,6H),1.71-1.59(m,5H),1.53-1.44( m,1H),1.38-1.21(m,4H),1.01-0.94(m,1H),0.88(t,J=7.3Hz,3H),0.39-0.32(m,4H)

[0518] MS m / z(ESI):864.0[(M+2H) / 2] + .

[0519] Example 25, N-((4S,12S,21S)-12-benzyl-21-(3-(2-(tert-butylsulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propionamido)-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolane[ 4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl-2,2-d2)-3,8,11,14,17,20-hexaoxo-5-oxa-2,7,10,13,16,19-hexaazapentacosan-25-yl)-2,5,8,11,14,17,20,23-octaoxahexacosan-26-amide (c-L2-1)

[0520] Step 1: Synthesis of 3-(2-(tert-butylsulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propanoic acid (Intermediate L4-1)

[0521] Under a nitrogen atmosphere, compound 8 (200 mg) was dissolved in anhydrous dichloromethane (10 mL) and cooled to -78°C. Trifluoroacetic acid (2.5 mL) was added at this temperature, and the mixture was slowly warmed to room temperature and stirred for 2 h. After the reaction, the solvent was removed under a stream of nitrogen, and the residue was washed with a 5:1 mixture of petroleum ether and ethyl acetate and filtered to obtain the title compound (150 mg).

[0522] MS m / z(ESI):342.1[M+H] + .

[0523] Step 2: Synthesis of resin-supported (32S,41S,49S)-41-benzyl-32-(3-(2-(tert-butylsulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propionamido)-49-cyclopropyl-26,33,36,39,42,45-hexaoxo-2,5,8,11,14,17,20,23,48-nonaoxa-27,34,37,40,43,46-hexaazapentacontan-50-oic acid (Intermediate L4-2)

[0524] Intermediate L2-3 (300 mg) was dissolved in DMF (5 mL), and intermediate L4-1 (106.67 mg), O-benzotriazole-tetramethyluronium hexafluorophosphate (178.20 mg), and diisopropylethylamine (91.09 mg) were added sequentially. The reaction solution was shaken at 25°C for 1 hour. After completion of the reaction, the resin was washed with methanol (10 mL) and dichloromethane (10 mL) three times, followed by filtration to dryness and drying under vacuum to obtain the title compound (300 mg).

[0525] MS m / z(ESI):583.0[(M+H) / 2] + .

[0526] Step 3: Synthesis of (32S,41S,49S)-41-benzyl-32-(3-(2-(tert-butylsulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propionamido)-49-cyclopropyl-26,33,36,39,42,45-hexaoxo-2,5,8,11,14,17,20,23,48-nonaoxa-27,34,37,40,43,46-hexaazapentacontan-50-oic acid (Intermediate L4-3)

[0527] Intermediate L4-2 (300 mg) was added to a mixed solvent of dichloromethane (10 mL) and 1,1,1,3,3,3-hexafluoroisopropanol (2.5 mL) and shaken at 25°C for 0.5 h. After the reaction, the reaction mixture was filtered to remove the resin, and the filtrate was concentrated to dryness under reduced pressure. The crude product was lyophilized to obtain the title compound (80 mg).

[0528] MS m / z(ESI):583.0[(M+H) / 2] + .

[0529] Step 4: N-((4S,12S,21S)-12-benzyl-21-(3-(2-(tert-butylsulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propionamido)-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolane[4, Synthesis of (5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl-2,2-d2)-3,8,11,14,17,20-hexaoxo-5-oxa-2,7,10,13,16,19-hexaazapentacosan-25-yl)-2,5,8,11,14,17,20,23-octaoxahexacosan-26-amide (c-L2-1)

[0530] Intermediate L4-3 (70 mg), intermediate L1-7 (24.57 mg), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (20.5 mg), 1-hydroxybenzotriazole (14.44 mg), and pyridine (12.68 mg) were dissolved in DMF (0.5 mL). The atmosphere was purged with nitrogen three times. The reaction mixture was stirred at 25°C under nitrogen for 1 hour. After completion of the reaction, the reaction mixture was directly purified by HPLC (column: C18 100×10 mm; mobile phase: [A: water (trifluoroacetic acid), B: acetonitrile]; B%: 18%-48%, 8 min) to obtain the title compound (6.3 mg).

[0531] 1H NMR (400MHz, DMSO-d6) δ = 9.25 (s, 1H), 8.69-8.64 (m, 1H), 8.60 (t, J = 6.4Hz, 1H), 8.32 ( t,J=5.6Hz,1H),8.25-8.16(m,1H),8.11(d,J=7.8Hz,1H),7.96(t,J=5.3Hz,1H),7.82 -7.75(m,1H),7.52(s,1H),7.29-7.19(m,5H),7.19-7.12(m,1H),6.50(s,1H),5.44(d ,J=8.8Hz,4H),4.86-4.71(m,2H),4.71-4.63(m,1H),4.55-4.47(m,1H),4.41-4.39(m, 1H),4.22-4.15(m,1H),3.81-3.60(m,9H),3.57(t,J=6.6Hz,3H),3.53-3.44(m,24H), 3.43-3.40(m,12H),3.24-3.20(m,4H),3.08-3.08(m,1H),3.08-2.94(m,2H),2.79-2.7 7(m,1H),2.28(t,J=6.5Hz,2H),1.93-1.79(m,2H),1.62(s,1H),1.48(d,J=9.3Hz,1H) ,1.37(s,9H),1.24(s,2H),1.02-0.93(m,1H),0.88(t,J=7.1Hz,3H),0.40-0.28(m,4H)

[0532] MS m / z(ESI):858.4[(M+2H) / 2] + .

[0533] Example 26, N-((4S,12S,25S)-12-benzyl-25-(3-(2-(tert-butylsulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propionamido)-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolane[4,5 -g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl-2,2-d2)-3,8,11,14,17,20,24-heptaoxo-5-oxa-2,7,10,13,16,19,23-heptaazanonacosan-29-yl)-2,5,8,11,14,17,20,23-octaoxahexacosan-26-amide (c-L5-1)

[0534] Step 1: Synthesis of resin-supported (15S,23S)-15-benzyl-23-cyclopropyl-1-(9H-fluoren-9-yl)-3,7,10,13,16,19-hexaoxo-2,22-dioxa-4,8,11,14,17,20-hexaazatetracosanoic acid (Intermediate L5-1)

[0535] Dissolve 3-(9H-fluoren-9-yl)methoxycarbonylaminopropionic acid (274.48 mg) in DMF (5 mL). Add L1-25 (total weight of substrate and resin: 500 mg, content: approximately 0.22 mmol), O-benzotriazole-tetramethyluronium hexafluorophosphate (336.58 mg), and diisopropylethylamine (257.02 mg) sequentially. Shake the reaction mixture on a shaker at 25°C for 1 hour. After the reaction, wash the resin with methanol (15 mL) and dichloromethane (15 mL) three times, filter the resin to dryness, and dry it under vacuum to obtain the title compound (528.4 mg).

[0536] MS m / z(ESI):779.5([M+Na)] + .

[0537] Step 2: Synthesis of resin-supported (2S,10S)-20-amino-10-benzyl-2-cyclopropyl-6,9,12,15,18-pentaoxo-3-oxa-5,8,11,14,17-pentaazaeicosane-1-carboxylic acid (Intermediate L5-2)

[0538] Intermediate L5-1 (528.4 mg) was dissolved in DMF (5 mL) and piperidine (42.97 mg). The reaction mixture was shaken at 25°C for 1 hour. After the reaction, the resin was washed with methanol (15 mL) and dichloromethane (15 mL) three times. The resin was then filtered to dryness and dried under vacuum to obtain the title compound (480.6 mg).

[0539] MS m / z(ESI):535.1([M+H)] + .

[0540] Step 3: Synthesis of resin-supported (32S,45S,53S)-32-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-45-benzyl-53-cyclopropyl-26,33,37,40,43,46,49-heptaoxo-2,5,8,11,14,17,20,23,52-nonaoxa-27,34,38,41,44,47,50-heptaazatetradecane-54-carboxylic acid (Intermediate L5-3)

[0541] Intermediate L2-3 (590.82 mg) was dissolved in DMF (5 mL), and compound L5-2 (480.6 mg), O-benzotriazole-tetramethyluronium hexafluorophosphate (295.66 mg), and diisopropylethylamine (225.77 mg) were added sequentially. The reaction mixture was shaken at 25°C for 1 hour. After the reaction, the resin was washed with methanol (15 mL) and dichloromethane (15 mL) three times. The resin was then filtered to dryness and dried under vacuum to obtain the title compound (502 mg).

[0542] Step 4: Synthesis of resin-supported (32S,45S,53S)-32-amino-45-benzyl-53-cyclopropyl-26,33,37,40,43,46,49-heptaoxo-2,5,8,11,14,17,20,23,52-nonaoxa-27,34,38,41,44,47,50-heptaazatetradecane-54-carboxylic acid (Intermediate L5-4)

[0543] Intermediate L5-3 (502 mg) was dissolved in DMF (5 mL) and piperidine (26.98 mg). The reaction mixture was shaken at 25°C for 1 hour. After the reaction, the resin was washed with methanol (15 mL) and dichloromethane (15 mL) three times. The resin was then filtered to dryness and dried under vacuum to obtain the title compound (468.5 mg).

[0544] Step 5: Synthesis of resin-supported methyl (32S,45S,53S)-45-benzyl-32-(3-(2-(tert-butylsulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propionamido)-53-cyclopropyl-26,33,37,40,43,46,49-heptaoxo-2,5,8,11,14,17,20,23,52-nonaoxa-27,34,38,41,44,47,50-heptaazatetracosan-54-carboxylic acid (Intermediate L5-5)

[0545] Intermediate L5-4 (220.0 mg) was dissolved in DMF (5 mL), followed by Intermediate L4-1 (74.10 mg), O-benzotriazole-tetramethyluronium hexafluorophosphate (82.87 mg), and diisopropylethylamine (63.28 mg). The reaction mixture was shaken at 25°C for 1 hour. After the reaction, the resin was washed with methanol (10 mL) and dichloromethane (10 mL) three times, followed by filtration to dryness and drying under vacuum to obtain the title compound (220 mg).

[0546] MS m / z(ESI):713.0([M+Na)] + .

[0547] Step 6: Synthesis of (32S,45S,53S)-45-benzyl-32-(3-(2-(tert-butylsulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propionamido)-53-cyclopropyl-26,33,37,40,43,46,49-heptaoxo-2,5,8,11,14,17,20,23,52-nonaoxa-27,34,38,41,44,47,50-heptaazatetracontane-54-carboxylic acid (Intermediate L5-6)

[0548] Intermediate L5-5 (220.00 mg) was added to a mixed solvent of dichloromethane (16 mL) and 1,1,1,3,3,3-hexafluoroisopropanol (4 mL) and shaken at 25°C for 15 minutes. After the reaction, the reaction mixture was filtered to remove the resin, and the filtrate was concentrated to dryness under reduced pressure to obtain the title compound (30 mg).

[0549] MS m / z(ESI):691.0[(M+2H) / 2] + .

[0550] Step 7: N-((4S,12S,25S)-12-benzyl-25-(3-(2-(tert-butylsulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propionamido)-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolane[4,5-g Synthesis of 14-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-2,2-d2)-3,8,11,14,17,20,24-heptaoxo-5-oxa-2,7,10,13,16,19,23-heptaazanonacosan-29-yl)-2,5,8,11,14,17,20,23-octaoxahexacosan-26-amide (c-L5-1)

[0551] Intermediate L5-5 (30 mg), intermediate L1-7 (17.49 mg), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (8.33 mg), 1-hydroxybenzotriazole (5.87 mg), and pyridine (5.16 mg) were dissolved in DMF (0.5 mL). The atmosphere was purged with nitrogen three times. The reaction mixture was stirred at 25°C under nitrogen for 1 hour. After completion of the reaction, the reaction mixture was directly purified by HPLC (column: C18 150×30mm×5μm; mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 25%-55%, 11 min) to obtain the title compound (7.1 mg).

[0552] MS m / z(ESI):893.7[(M+2H) / 2] + .

[0553] 1 H NMR (400MHz, DMSO-d6) δ = 9.25 (s, 1H), 8.67 (s, 1H), 8.63-8.57 (m, 1H), 8.30 (s, 1H), 8.22-8.09(m,2H),8.03(s,1H),7.96(s,1H),7.83-7.75(m,2H),7.52(s,1H),7.28- 7.19(m,5H),7.17(d,J=6.6Hz,1H),6.50(s,1H),5.44(d,J=9.0Hz,4H),4.85-4.71( m,2H),4.68-4.66(m,1H),4.51(s,1H),4.41-4.39(m,1H),4.18-4.09(m,1H),4.06(s ,1H),3.77-3.65(m,12H),3.61-3.55(m,9H),3.51-3.49(m,22H),3.24(s,5H),3.04 (d,J=9.8Hz,1H),2.97(d,J=6.1Hz,2H),2.79-2.77(m,1H),2.32-2.24(m,3H),1.93- 1.80(m,2H),1.56(s,2H),1.45(d,J=8.5Hz,2H),1.37(s,9H),1.34-1.28(m,2H),1. 22(d,J=15.6Hz,3H),0.96(d,J=6.8Hz,1H),0.87(t,J=7.3Hz,3H),0.40-0.30(m,4H)

[0554] Example 27, N-((4S,12S)-12-benzyl-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolane-[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl-2,2-d2)-3,8,11, 14,17-pentaoxo-5-oxa-2,7,10,13,16-pentaazaoctadec-18-yl)-1-(3-(2-(tert-butylsulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propionamido)-3,6,9,12,15,18,21,24-octaoxaheptacosane-27-amide (c-L6-1)

[0555] Step 1: Synthesis of resin-supported (39S,47S)-39-benzyl-47-cyclopropyl-1-(9H-fluoren-9-yl)-3,31,34,37,40,43-hexaoxo-2,7,10,13,16,19,22,25,28,46-decaoxa-4,32,35,38,41,44-hexaazatetraoctadecane-48-oic acid (Intermediate L6-1)

[0556] Dissolve 1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13,16,19,22,25,28-nonaoxa-4-azatrionecontane-31-carboxylic acid (468.16 mg) in DMF (5 mL). Add intermediate L1-25 (400.0 mg), O-benzotriazole-tetramethyluronium hexafluorophosphate (269.26 mg), and diisopropylethylamine (205.61 mg) sequentially. Shake the reaction mixture on a shaker at 25°C for 1 hour. After completion of the reaction, wash the resin with methanol (15 mL) and dichloromethane (15 mL) three times, filter to dryness, and dry under vacuum to obtain the title compound (450 mg).

[0557] MS m / z(ESI):1109.4([M+H)] + .

[0558] Step 2: Synthesis of resin-supported (2S,10S)-4,4-amino-10-benzyl-2-cyclopropyl-6,9,12,15,18-pentaoxo-3,21,24,27,30,33,36,39,42-nonaoxa-5,8,11,14,17-pentaazatetradecanoic acid (Compound L6-2)

[0559] Intermediate L6-1 (440 mg) was dissolved in DMF (10 mL) and piperidine (2 mL). The reaction mixture was shaken at 25°C for 1 hour. After the reaction, the resin was washed with methanol (15 mL) and dichloromethane (15 mL) three times. The resin was then filtered to dryness and dried under vacuum to obtain the title compound (365 mg).

[0560] MS m / z(ESI):887.5([M+H)] + .

[0561] Step 3: Synthesis of resin-supported (2S,10S)-10-benzyl-48-(2-(tert-butylsulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)-2-cyclopropyl-6,9,12,15,18,46-hexaoxo-3,21,24,27,30,33,36,39,42-nonaoxa-5,8,11,14,17,45-hexaazatetraoctadecanoic acid (Intermediate L6-3)

[0562] Intermediate L6-2 (365.0 mg) was dissolved in DMF (5 mL), and intermediate L4-1 (140.71 mg), O-benzotriazole-tetramethyluronium hexafluorophosphate (157.35 mg), and diisopropylethylamine (120.16 mg) were added sequentially. The reaction mixture was shaken at 25°C for 1 hour. After completion of the reaction, the resin was washed with methanol (10 mL) and dichloromethane (10 mL) three times, followed by filtration to dryness and drying under vacuum to obtain the title compound (285 mg).

[0563] MS m / z(ESI):1210.4([M+H)] + .

[0564] Step 4: Synthesis of (2S,10S)-10-benzyl-48-(2-(tert-butylsulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)-2-cyclopropyl-6,9,12,15,18,46-hexaoxo-3,21,24,27,30,33,36,39,42-nonaoxa-5,8,11,14,17,45-hexaazatetraoctadecanoic acid (Intermediate L6-4)

[0565] Intermediate L6-3 (380.00 mg) was added to a mixed solvent of dichloromethane (8 mL) and 1,1,1,3,3,3-hexafluoroisopropanol (2 mL) and shaken at 25°C for 15 minutes. After the reaction, the reaction mixture was filtered to remove the resin, and the filtrate was concentrated to dryness under reduced pressure to obtain the title compound (110 mg).

[0566] MS m / z(ESI):606.0[(M+2H) / 2] + .

[0567] Step 5: N-((4S,12S)-12-benzyl-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl-2,2-d2)-3,8,11,14 Synthesis of 1-(3-(2-(tert-butylsulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propionamido)-3,6,9,12,15,18,21,24-octaoxaheptacosane-27-amide (c-L6-1)

[0568] Intermediate L6-4 (50 mg), intermediate L1-7 (17.49 mg), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (15.84 mg), 1-hydroxybenzotriazole (11.16 mg), and pyridine (9.80 mg) were dissolved in DMF (0.5 mL). The atmosphere was purged with nitrogen three times. The reaction mixture was stirred at 25°C under nitrogen for 1 hour. After completion of the reaction, the reaction mixture was directly purified by HPLC (column: C18 150×30mm×5μm; mobile phase: [Phase A: water (0.225% formic acid), Phase B: acetonitrile]; B%: 25%-55%, 9 min) to obtain the title compound (14.1 mg).

[0569] MS m / z(ESI):808.6[(M+2H) / 2] + .

[0570] 1H NMR (400MHz, DMSO-d6) δ = 9.26 (s, 1H), 8.69-8.56 (m, 2H), 8.32-8.25 (m, 1H), 8.19-8.14 (m, 1H), 8.13-8.04 (m, 2H), 8.02-7.97 (m, 1H), 7. 80(s,1H),7.52(s,1H),7.28-7.21(m,5H),7.19-7.17(m,1H),6.50(s,1H),5.44(d,J=8.3Hz,4H),4.79(d,J=7.8Hz,2H),4.68-4.66(m,1H ),4.54-4.46(m,1H),4.41-4.39(m,1H),3.77-3.66(m,10H),3.62-3.57(m,3H),3.51-3.46(m,30H),3.22(d,J=6.8Hz,4H),3.09-3.01(m ,1H),2.80-2.78(m,1H),2.46-2.37(m,4H),1.93-1.80(m,2H),1.37(s,9H),1.01-0.94(m,1H),0.88(t,J=7.3Hz,3H),0.39-0.31(m,4H).

[0571] Example 28, N-((4S,12S,21S)-12-benzyl-21-(3-(2-(tert-butylsulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propionamido)-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-8,10,11,13-tetrahydro-7H-yl)-1,3 ]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl-2,2-d2)-3,8,11,14,17,20-hexaoxo-5-oxa-2,7,10,13,16,19-hexaazapentacosan-25-yl)-2,5,8,11-tetraoxatetradecane-14-amide (c-L7-1)

[0572] Step 1: Synthesis of (S)-20-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-14-oxo-2,5,8,11-tetraoxa-15-azaheneicosane-21-oic acid (Intermediate L7-1)

[0573] (((9H-fluoren-9-yl)methoxy)carbonyl)-L-lysine (2.1 g) and O-(2,5-dioxopyrrolidin-1-yl)-2,5,8,11-tetraoxatetradecanoate-14-oate (1.9 g) were dissolved in DMF (40 mL). The atmosphere was purged with nitrogen three times, and the reaction was stirred at 25°C for 16 h. After completion of the reaction, the crude product was separated by column chromatography (ethyl acetate / petroleum ether, 0-80%) to obtain the title compound (2.3 g).

[0574] MS m / z(ESI):587.1[M+H] + .

[0575] Step 2: Synthesis of resin-supported (20S,29S,37S)-20-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-29-benzyl-37-cyclopropyl-14,21,24,27,30,33-hexaoxo-2,5,8,11,36-pentaoxa-15,22,25,28,31,34-hexaazatriacontanoic acid (Intermediate L7-2)

[0576] Intermediate L7-1 (517.24 mg) was dissolved in DMF (5 mL). Intermediate L1-25 (total weight of loaded substrate and resin: 500 mg, content: approximately 0.22 mmol), O-benzotriazole-tetramethyluronium hexafluorophosphate (336.58 mg), and diisopropylethylamine (257.02 mg) were added sequentially. The reaction mixture was shaken at 25°C for 1 hour. After completion of the reaction, the resin was washed with methanol (15 mL) and dichloromethane (15 mL) three times, followed by filtration to dryness and drying under vacuum to obtain the title compound (555 mg).

[0577] MS m / z(ESI):1032.3([M+H)] + .

[0578] Step 3: Synthesis of resin-supported (20S,29S,37S)-20-amino-29-benzyl-37-cyclopropyl-14,21,24,27,30,33-hexaoxo-2,5,8,11,36-pentaoxa-15,22,25,28,31,34-hexaazaoctatriacont-38-oic acid (Intermediate L7-3)

[0579] Intermediate L7-2 (555 mg) was dissolved in DMF (5 mL) and piperidine (35.72 mg). The reaction mixture was shaken at 25°C for 1 hour. After the reaction, the resin was washed with methanol (15 mL) and dichloromethane (15 mL) three times. The resin was then filtered to dryness and dried under vacuum to obtain the title compound (428 mg).

[0580] MS m / z(ESI):810.2([M+H)] + .

[0581] Step 4: Synthesis of resin-supported (20S,29S,37S)-29-benzyl-20-(3-(2-(tert-butylsulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propionamido)-37-cyclopropyl-14,21,24,27,30,33-hexaoxo-2,5,8,11,36-pentaoxa-15,22,25,28,31,34-hexaazaoctatriacontanoic acid (Intermediate L7-4)

[0582] Intermediate L7-3 (428 mg) was dissolved in DMF (5 mL), and intermediate L4-1 (176.55 mg), O-benzotriazole-tetramethyluronium hexafluorophosphate (197.43 mg), and diisopropylethylamine (150.76 mg) were added sequentially. The reaction mixture was shaken at 25°C for 1 hour. After completion of the reaction, the resin was washed with methanol (10 mL) and dichloromethane (10 mL) three times, followed by filtration to dryness and drying under vacuum to obtain the title compound (438 mg).

[0583] MS m / z(ESI):1155.3([M+Na)] + .

[0584] Step 5: Synthesis of (20S,29S,37S)-29-benzyl-20-(3-(2-(tert-butylsulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propionamido)-37-cyclopropyl-14,21,24,27,30,33-hexaoxo-2,5,8,11,36-pentaoxa-15,22,25,28,31,34-hexaazaoctatriacontanoic acid (Intermediate L7-5)

[0585] Intermediate L7-4 (438 mg) was added to a mixed solvent of dichloromethane (8 mL) and 1,1,1,3,3,3-hexafluoroisopropanol (2 mL) and shaken at 25°C for 15 minutes. After the reaction, the reaction mixture was filtered to remove the resin, and the filtrate was concentrated to dryness under reduced pressure to obtain the title compound (50 mg).

[0586] MS m / z(ESI):1155.3([M+Na)] + .

[0587] Step 6: N-((4S,12S,21S)-12-benzyl-21-(3-(2-(tert-butylsulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propionamido)-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-8,10,11,13-tetrahydro-7H-oxo[1,3]dihydro- Synthesis of oxalo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl-2,2-d2)-3,8,11,14,17,20-hexaoxo-5-oxa-2,7,10,13,16,19-hexaazapentacosan-25-yl)-2,5,8,11-tetraoxatetradecane-14-amide (c-L7-1)

[0588] Intermediate L7-5 (50 mg), intermediate L1-7 (18.68 mg), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (16.92 mg), 1-hydroxybenzotriazole (11.92 mg), and pyridine (10.47 mg) were dissolved in DMF (1 mL). The atmosphere was purged with nitrogen three times. The reaction mixture was stirred at 25°C under nitrogen for 1 hour. After completion of the reaction, the reaction mixture was directly purified by HPLC (column: Boston Prime C18 100*10 mm; mobile phase: [phase A: water (0.225% formic acid), phase B: acetonitrile]; B%: 20%-42%, 15 min) to obtain the title compound (5.7 mg).

[0589] MS m / z(ESI):770.0[(M+2H) / 2] + .

[0590] 1H NMR (400MHz, DMSO-d6) δ = 9.23 (s, 1H), 8.76-8.64 (m, 1H), 8.59 (t, J = 6.4Hz, 1H ),8.31(t,J=5.6Hz,1H),8.28-8.14(m,2H),8.14-8.04(m,1H),7.96(t,J=5.2 Hz,1H),7.86-7.69(m,2H),7.51(s,1H),7.28-7.19(m,4H),7.16(J=2.6,5.7H z,1H),6.63-6.51(m,1H),6.50(s,1H),5.43(d,J=7.5Hz,3H),4.90-4.57(m,3H ),4.50(J=5.1,8.5Hz,1H),4.39(J=6.5,9.9Hz,1H),4.23-4.12(m,1H),3.81- 3.44(m,27H),3.22(s,3H),3.08-2.91(m,4H),2.86-2.72(m,2H),2.27(t,J=6 .4Hz,2H),1.96-1.78(m,2H),1.67-1.56(m,1H),1.53-1.44(m,1H),1.43-1.2 7(m,9H),1.23(s,3H),0.99-0.91(m,1H),0.91-0.81(m,3H),0.43-0.26(m,4H)

[0591] Example 29, N-((4S,12S,21S)-12-benzyl-21-(3-(2-(tert-butylsulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propionamido)-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolane[4,5-g] Pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl-2,2-d2)-3,8,11,14,17,20-hexaoxo-5-oxa-2,7,10,13,16,19-hexaazapentacosan-25-yl)-2,5,8,11,14,17,20,23,26,29,32,35-dodecatriazol-38-amide (c-L8-1)

[0592] Step 1: Synthesis of (S)-44-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-38-oxo-2,5,8,11,14,17,20,23,26,29,32,35-dodeca-39-azapentatetradecan-45-oic acid (Intermediate L8-1)

[0593] (((9H-fluoren-9-yl)methoxy)carbonyl)-L-lysine (800 mg) and O-(2,5-dioxopyrrolidin-1-yl)-2,5,8,11,14,17,20,23,26,29,32,35-dodeca-triadecanoate (429 mg) were dissolved in DMF (4 mL), and the atmosphere was replaced with nitrogen three times. The reaction was stirred at 25 ° C for 16 h. After the reaction was completed, the crude product was purified by reverse phase high performance liquid chromatography ( 80g C18 column, mobile phase gradient 0-50% acetonitrile / water, flow rate 40mL / min) to obtain the title compound (650mg).

[0594] MS m / z(ESI):940.0[M+H] + .

[0595] Step 2: Synthesis of resin-supported (44S,53S,61S)-44-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-53-benzyl-61-cyclopropyl-38,45,48,51,54,57-hexaoxo-2,5,8,11,14,17,20,23,26,29,32,35,60-trideca-39,46,49,52,55,58-hexaazahexadodecane-62-oic acid (Intermediate L8-2)

[0596] Intermediate L8-1 (650 mg) was added to DMF (10 mL), followed by Intermediate L1-25 (400 mg), O-benzotriazole-tetramethyluronium hexafluorophosphate (336.58 mg), and diisopropylethylamine (205.62 mg). The reaction mixture was nitrogen-purged three times and shaken on a shaker at 25°C for 1 h. After completion of the reaction, the resin was washed three times with methanol (10 mL) and then with dichloromethane (10 mL). The resin was then filtered to dryness and dried under vacuum to yield the title compound (430 mg).

[0597] Step 3: Synthesis of resin-supported (44S,53S,61S)-44-amino-53-benzyl-61-cyclopropyl-38,45,48,51,54,57-hexaoxo-2,5,8,11,14,17,20,23,26,29,32,35,60-trideca-39,46,49,52,55,58-hexaazahexadodecane-62-oic acid (Intermediate L8-3)

[0598] Intermediate L8-2 (430 mg) was dissolved in DMF (5 mL) and piperidine (1.08 g, 1.25 mL) was added. The reaction mixture was shaken at 25°C for 1 hour. After the reaction, the resin was washed with methanol (10 mL) and dichloromethane (10 mL) three times, followed by filtration to dryness and drying under vacuum to obtain the title compound (410 mg).

[0599] Step 4: Synthesis of resin-supported (44S,53S,61S)-53-benzyl-44-(3-(2-(tert-butylsulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propionamido)-61-cyclopropyl-38,45,48,51,54,57-hexaoxo-2,5,8,11,14,17,20,23,26,29,32,35,60-trideca-39,46,49,52,55,58-hexaazahexadodecane-62-oic acid (Intermediate L8-4)

[0600] Intermediate L8-3 (410 mg) and intermediate L4-1 (144.48 mg) were dissolved in DMF (10 mL). O-benzotriazole-tetramethyluronium hexafluorophosphate (214.89 mg) and diisopropylethylamine (182.33 mg) were added. The reaction mixture was shaken at 25°C for 1 hour. After the reaction, the resin was washed with methanol (10 mL) and dichloromethane (10 mL) three times, followed by filtration to dryness. The resin was then dried under vacuum to obtain the title compound (420 mg).

[0601] Step 5: Synthesis of (44S,53S,61S)-53-benzyl-44-(3-(2-(tert-butylsulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propionamido)-61-cyclopropyl-38,45,48,51,54,57-hexaoxo-2,5,8,11,14,17,20,23,26,29,32,35,60-trideca-39,46,49,52,55,58-hexaazahexadodecane-62-oic acid (Intermediate L8-5)

[0602] Intermediate L8-4 (420 mg) was added to a mixed solvent of dichloromethane (8 mL) and 1,1,1,3,3,3-hexafluoroisopropanol (2 mL) and shaken at 25°C for 0.5 h. After the reaction, the reaction mixture was filtered to remove the resin, and the filtrate was concentrated to dryness under reduced pressure to obtain the title compound (130 mg).

[0603] Step 6: N-((4S,12S,21S)-12-benzyl-21-(3-(2-(tert-butylsulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propionamido)-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolano[4,5-g]pyrano[1,3]dioxolano[4,5-g]pyrano[1,3]dioxolano[4,5-g]pyrano[1,3]dioxolano[4,5-g]pyrano[1,3]dioxolano[4,5-g]pyrano[1,3]dioxolano[4,8-d]pyrano[1,8]dioxolano[4,5-g]pyrano[1,3]dioxolano[4,8-d]pyrano[1,8]dioxolano[4,5-g]pyrano[1,3]dioxolano[4,8-d]pyrano[1,8]dioxolano[4,5-g]pyrano[1,8]dioxolano[4,8-d ... Synthesis of [3',4':6,7]indolizino[1,2-b]quinolin-14-yl-2,2-d2)-3,8,11,14,17,20-hexaoxo-5-oxa-2,7,10,13,16,19-hexaazapentacosan-25-yl)-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoctatriacontane-38-amide (c-L8-1)

[0604] Intermediate L8-5 (70 mg), intermediate L1-7 (21.94 mg), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (18.20 mg), pyridine (11.26 mg), and 1-hydroxybenzotriazole (12.83 mg) were dissolved in DMF (2 mL). The atmosphere was purged with nitrogen three times. The reaction mixture was stirred at 25°C under nitrogen for 2 h. After completion of the reaction, the reaction mixture was directly purified by HPLC (column: C18 100*10 mm; mobile phase: [phase A: water (0.225% formic acid), phase B: acetonitrile]; B%: 28%-35%, 20 min) to obtain the title compound (5.1 mg).

[0605] 1H NMR (400MHz, DMSO-d6) δ = 9.27-9.23 (m, 1H), 8.70 (d, J = 5.3Hz, 1H), 8.68-8.61 (m, 1H),8.40(s,2H),8.28-8.23(m,1H),8.22-8.13(m,1H),8.04-7.97(m,1H),7.84- 7.76(m,2H),7.54-7.50(m,1H),7.26(s,1H),7.23-7.21(m,4H),7.19-7.12(m,1H ),6.61-6.44(m,1H),5.46-5.40(m,4H),4.86-4.72(m,2H),4.71-4.63(m,2H),4.5 7-4.46(m,2H),4.46-4.35(m,2H),4.25-4.14(m,2H),3.82-3.55(m,28H),3.49-3 .37(m,22H),3.28-3.18(m,9H),3.10-2.95(m,5H),2.85-2.74(m,2H),2.28(t,J= 6.5Hz,2H),1.92-1.80(m,2H),1.66-1.59(m,1H),1.51-1.42(m,1H),1.42-1.30( m,9H),1.28-1.16(m,2H),1.00-0.95(m,1H),0.89-0.85(m,3H),0.38-0.32(m,4H)

[0606] MS m / z(ESI):946.5[M / 2+H] + .

[0607] Example 30, N-((4S,12S)-12-benzyl-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolane-[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl-2,2-d2)- 3,8,11,14,17-pentaoxo-5-oxa-2,7,10,13,16-pentaazaoctadec-18-yl)-1-(3-(2-(tert-butylsulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propionamido)-3,6,9,12-tetraoxapentadecan-15-amide (c-L9-1)

[0608] Step 1: Synthesis of resin-supported (27S,35S)-27-benzyl-35-cyclopropyl-1-(9H-fluoren-9-yl)-3,19,22,25,28,31-hexaoxo-2,7,10,13,16,34-hexaoxa-4,20,23,26,29,32-hexaazahexatriacontanoic acid (Intermediate L9-1)

[0609] To a mixture of 1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13,16-pentaoxa-4-azanonadecane-19-carboxylic acid (420 mg), intermediate L1-25 (total weight of loaded substrate and resin 500 mg, content approximately 0.22 mmol), N,N-diisopropylethylamine (257 mg), and benzotriazole-N,N,N,N-tetramethyluronium hexafluorophosphate (497 mg) was added DMF (5 mL) at room temperature under nitrogen. The resulting mixture was shaken on a shaker at 20°C for 1 hour. The resin was then washed with methanol (10 mL) and dichloromethane (10 mL) three times, filtered to dryness, and dried under vacuum to obtain the title compound (550 mg).

[0610] Step 2: Synthesis of resin-supported (2S,10S)-32-amino-10-benzyl-2-cyclopropyl-6,9,12,15,18-pentaoxo-3,21,24,27,30-pentaoxa-5,8,11,14,17-pentaazatriacontanoic acid (Intermediate L9-2)

[0611] To a solution of intermediate L9-1 (550 mg) in DMF (5 mL) was added piperidine (500 μL) at room temperature under nitrogen. The resulting mixture was shaken on a shaker at 20°C for 1 h. The resin was then washed three times with methanol (10 mL) and dichloromethane (10 mL). The resin was then filtered to dryness and dried under vacuum to obtain the title compound (500 mg).

[0612] LC-MS: m / z(ESI):733.2[M+Na] + .

[0613] Step 3: Synthesis of resin-supported (2S,10S)-10-benzyl-36-(2-(tert-butylsulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)-2-cyclopropyl-6,9,12,15,18,34-hexaoxo-3,21,24,27,30-pentaoxa-5,8,11,14,17,33-hexaazahexatriacontanoic acid (Intermediate L9-3)

[0614] Under a nitrogen atmosphere, DMF (5 mL) was added to a mixture of Intermediate L9-2 (500 mg), Intermediate L4-1 (187 mg), N,N-diisopropylethylamine (193 mg), and benzotriazole-N,N,N,N-tetramethyluronium hexafluorophosphate (253 mg). The resulting mixture was shaken on a shaker at 20°C for 1 hour. The resin was then washed with methanol (10 mL) and dichloromethane (10 mL) three times, filtered to dryness, and dried under vacuum to obtain the title compound (500 mg).

[0615] Step 4: Synthesis of (2S,10S)-10-benzyl-36-(2-(tert-butylsulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)-2-cyclopropyl-6,9,12,15,18,34-hexaoxo-3,21,24,27,30-pentaoxa-5,8,11,14,17,33-hexaazahexatriacontanoic acid (Intermediate L9-4)

[0616] Under a nitrogen atmosphere, intermediate L9-3 (500 mg) was dissolved in dichloromethane (16 mL) and 1,1,1,3,3,3-hexafluoroisopropanol (4 mL). The resulting mixture was shaken at 20°C for 1 hour. The reaction mixture was then filtered to remove the resin, and the filtrate was concentrated to dryness under reduced pressure. Acetonitrile (4 mL) and purified water (8 mL) were added and lyophilized in a freeze dryer to provide the title compound (150 mg).

[0617] Step 5: N-((4S,12S)-12-benzyl-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl-2,2-d2)-3,8 Synthesis of 1-(3-(2-(tert-butylsulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propionamido)-3,6,9,12-tetraoxapentadecan-15-amide (c-L9-1)

[0618] Under a nitrogen atmosphere, DMF (2 mL) was added to a mixture of Intermediate L9-4 (100 mg), Intermediate L1-7 (45.0 mg), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (37.1 mg), 1-hydroxybenzotriazole (26.1 mg), and pyridine (23.0 mg). The resulting mixture was stirred at room temperature for 1 hour. The reaction mixture was then directly purified by HPLC (column: C18 100×10 mm; mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 15%-40%, 18 min) to obtain the title compound (10.6 mg).

[0619] LC-MS: m / z(ESI):1439.4[M+H] +

[0620] 1 H NMR (400MHz, DMSO-d6) δ9.29-9.24(m,1H),8.60(t,J=6.4Hz,2H),8.33-8.26(m,1H),8.21-8.13(m,1H),8.13-8.04(m,2H),8.00(t,J=5.5Hz,1H ),7.80(s,1H),7.52(s,1H),7.26-7.22(m,5H),7.19-7.14(m,1H),6.50 (s,1H),5.44(d,J=8.4Hz,4H),4.83-4.72(m,2H),4.70-4.63(m,1H),4. 55-4.47(m,1H),4.45-4.37(m,1H),3.77-3.67(m,10H),3.61-3.55(m,3 H),3.50-3.45(m,14H),3.26-3.18(m,4H),3.05-3.03(m,1H),2.84-2.7 4(m,1H),2.46-2.35(m,4H),1.92-1.81(m,2H),1.41-1.33(m,9H),1.00 -0.94(m,1H),0.90-0.86(m,3H),0.39-0.36(m,2H),0.34-0.30(m,2H).

[0621] Example 31, N-((4S,12S)-12-benzyl-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-[1,3]dioxolane-[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl-2,2-d2)-3,8,11,14,17-pentaoxo -5-oxa-2,7,10,13,16-pentaazaoctadec-18-yl)-1-(3-(2-(tert-butylsulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propionamido)-3,6,9,12,15,18,21,24,27,30,33,36-dodeca-nonatriacontane-39-amide (c-L10-1)

[0622] Step 1: Synthesis of (51S,59S)-51-benzyl-59-cyclopropyl-1-(9H-fluoren-9-yl)-3,43,46,49,52,55-hexaoxo-2,7,10,13,16,19,22,25,28,31,34,37,40,58-tetradecanoic acid-4,44,47,50,53,56-hexaazahexadecanoic acid (Intermediate L10-1)

[0623] Intermediate L1-25 (total weight of loaded substrate and resin: 500 mg, content: approximately 0.22 mmol) was dissolved in DMF (5 mL). 1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13,16,19,22,25,28,31,34,37,40-trideca-4-azatricatetradecanoic acid (723.85 mg), O-benzotriazole-tetramethyluronium hexafluorophosphate (336.58 mg), and diisopropylethylamine (257.02 mg) were added sequentially. The reaction mixture was shaken at 25°C for 1 hour. After completion of the reaction, the resin was washed with methanol (10 mL) and dichloromethane (10 mL) three times, filtered to dryness, and dried under vacuum to obtain the title compound (500 mg).

[0624] MS m / z(ESI):1307.4[M+Na] + .

[0625] Step 2: Synthesis of resin-supported (2S,10S)-56-amino-10-benzyl-2-cyclopropyl-6,9,12,15,18-pentaoxo-3,21,24,27,30,33,36,39,42,45,48,51,54-trideca-5,8,11,14,17-pentaazahexadecanoic acid (Intermediate L10-2)

[0626] Intermediate L10-1 (500 mg) was dissolved in DMF (2 mL) and piperidine (0.5 mL) was added. The reaction mixture was shaken at 25°C for 1 hour. After the reaction, the resin was washed with methanol (10 mL) and dichloromethane (10 mL) three times, followed by filtration to dryness and drying under vacuum to obtain the title compound (470 mg).

[0627] Step 3: Synthesis of resin-supported (2S,10S)-10-benzyl-60-(2-(tert-butylsulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)-2-cyclopropyl-6,9,12,15,18,58-hexaoxo-3,21,24,27,30,33,36,39,42,45,48,51,54-trideca-5,8,11,14,17,57-hexaazahexadecanoic acid (Intermediate L10-3)

[0628] Intermediate L10-2 (470 mg) was dissolved in DMF (5 mL), and intermediate L4-1 (192.54 mg), O-benzotriazole-tetramethyluronium hexafluorophosphate (176.25 mg), and diisopropylethylamine (134.59 mg) were added sequentially. The reaction mixture was shaken at 25°C for 1 hour. After completion of the reaction, the resin was washed with methanol (10 mL) and dichloromethane (10 mL) three times, followed by filtration to dryness and drying under vacuum to obtain the title compound (460 mg).

[0629] Step 4: Synthesis of (2S,10S)-10-benzyl-60-(2-(tert-butylsulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)-2-cyclopropyl-6,9,12,15,18,58-hexaoxo-3,21,24,27,30,33,36,39,42,45,48,51,54-trideca-5,8,11,14,17,57-hexaazahexadecanoic acid (Intermediate L10-4)

[0630] Intermediate L10-3 (460 mg) was added to a mixed solvent of dichloromethane (4 mL) and 1,1,1,3,3,3-hexafluoroisopropanol (1 mL) and shaken at 25°C for 0.5 h. After the reaction, the reaction mixture was filtered to remove the resin, and the filtrate was concentrated under reduced pressure and lyophilized to obtain the title compound (130 mg).

[0631] Step 5: N-((4S,12S)-12-benzyl-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl-2,2-d2)-3,8,11,14,17-pentaoxo-5-(4-hydroxy-1-(4-hydroxy-1-(4-hydroxy-1-(4-hydroxy-1-(4-hydroxy-1-(4-hydroxy-1-(4-hydroxy-1-(4-hydroxy-1-(4-hydroxy-1-(4-hydroxy-1-(4-hydroxy-1-(4-hydroxy-1-(4-hydroxy-1-(4-hydroxy-1-(4-hydroxy-1-(4-hydroxy-1-(4-hydroxy-1-(4-hydroxy-1- Synthesis of 1-(3-(2-(tert-butylsulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propionamido)-3,6,9,12,15,18,21,24,27,30,33,36-dodeca-nonatriacontane-39-amide (c-L10-1)

[0632] Intermediate L10-4 (50 mg), intermediate L1-7 (16.80 mg), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (13.83 mg), pyridine (8.56 mg), and 1-hydroxybenzotriazole (9.75 mg) were dissolved in DMF (1 mL). The atmosphere was purged with nitrogen three times. The reaction mixture was stirred at 25°C under nitrogen for 2 h. After completion of the reaction, the reaction mixture was directly purified by HPLC (column: Boston Prime 100*10 mm; mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 20%-42%, 15 min) to obtain the title compound (3.3 mg).

[0633] 1H NMR(400MHz,DMSO-d6)δ9.25(s,1H),8.70-8.58(m,2H),8.34-8.26(m,1H),8.19-8.15(m,1H),8.15-8.04(m,2H),8.03-7.97(m,1H),7. 79(s,1H),7.51(s,1H),7.27-7.21(m,5H),7.19-7.15(m,1H),6.82-6.73(m,1H),6.50(s,1H),5.50-5.37(m,4H),4.84-4.70(m,2H),4.6 7-4.65(m,1H),4.51-4.45(m,1H),4.42-4.36(m,1H),3.76-3.66(m,10H),3.59(s,3H),3.50-3.46(m,45H),3.24-3.17(m,4H),3.06-3. 01(m,1H),2.82-2.75(m,1H),2.45-2.35(m,4H),1.92-1.80(m,2H),1.36(s,9H),0.93(s,1H),0.87(t,J=7.3Hz,3H),0.38-0.30(m,4H).

[0634] MS m / z(ESI):896.6[M / 2+H] + .

[0635] Example 32, N-((4S,12S)-12-benzyl-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolane-[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl-2,2-d2)-3,8,11 ,14,17-pentaoxo-5-oxa-2,7,10,13,16-pentaazaoctadec-18-yl)-1-(3-(2-(cyclopentanesulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propionamido)-3,6,9,12,15,18,21,24-octaoxaheptacosane-27-amide (b-L6-1)

[0636] Step 1: Synthesis of resin-supported (39S,47S)-39-benzyl-47-cyclopropyl-1-(9H-fluoren-9-yl)-3,31,34,37,40,43-hexaoxo-2,7,10,13,16,19,22,25,28,46-decaoxa-4,32,35,38,41,44-hexaazaoctadecane-48-carboxylic acid (Intermediate 2)

[0637] At room temperature under nitrogen, N,N-dimethylformamide (10 mL) was added to a mixture of Intermediate 1A (1.17 g), Intermediate L1-25 (total weight of loaded substrate and resin: 1 g, content: approximately 0.43 mmol), N,N-diisopropylethylamine (514.05 mg), and benzotriazole-N,N,N,N-tetramethyluronium hexafluorophosphate (1.01 g). The resulting mixture was shaken on a shaker at 20°C for 1 h. After the reaction, the resin was washed three times with methanol (20 mL) and then with dichloromethane (20 mL). The resin was then filtered to dryness and dried under vacuum to obtain the title compound (1.03 g).

[0638] Step 2: Synthesis of resin-supported (2S,10S)-4,4-amino-10-benzyl-2-cyclopropyl-6,9,12,15,18-pentaoxo-3,21,24,27,30,33,36,39,42-nonaoxa-5,8,11,14,17-pentaazatetradecanoic acid (L11-2)

[0639] To a solution of intermediate L11-1 (1.03 g) in N,N-dimethylformamide (10 mL) was added piperidine (2.5 mL) at room temperature under nitrogen, and the resulting mixture was shaken on a shaker at 20°C for 1 h. After the reaction, the resin was washed three times with methanol (20 mL) and then with dichloromethane (20 mL). The resin was then filtered to dryness and dried under vacuum to obtain the title compound (993 mg).

[0640] LC-MS: m / z(ESI):887.6[M+H] + .

[0641] Step 3: Synthesis of resin-supported (2S,10S)-10-benzyl-48-(2-(cyclopentanesulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)-2-cyclopropyl-6,9,12,15,18,46-hexaoxo-3,21,24,27,30,33,36,39,42-nonaoxa-5,8,11,14,17,45-hexaazatetraoctadecanoic acid (Intermediate L11-3)

[0642] Under a nitrogen atmosphere, N,N-dimethylformamide (15 mL) was added to a mixture of intermediate L11-2 (993 mg), intermediate L3-1 (396.28 mg), N,N-diisopropylethylamine (326.90 mg), and benzotriazole-N,N,N,N-tetramethyluronium hexafluorophosphate (639.49 mg). The resulting mixture was shaken on a shaker at 20°C for 1 hour. LCMS confirmed the complete reaction of the starting materials. After the reaction was complete, the resin was washed with methanol (20 mL) and dichloromethane (20 mL) three times, filtered to dryness, and dried under vacuum to obtain the title compound (1.02 g).

[0643] LC-MS: m / z(ESI):1245.2[M+Na] +

[0644] Step 4: Synthesis of (2S,10S)-10-benzyl-48-(2-(cyclopentanesulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)-2-cyclopropyl-6,9,12,15,18,46-hexaoxo-3,21,24,27,30,33,36,39,42-nonaoxa-5,8,11,14,17,45-hexaazatetraoctadecanoic acid (Intermediate L11-4)

[0645] Under a nitrogen atmosphere, intermediate L11-3 (1.02 g) was dissolved in dichloromethane (16 mL) and 1,1,1,3,3,3-hexafluoroisopropanol (4 mL). The resulting mixture was shaken at 20°C for 1 hour. After the reaction, the reaction mixture was filtered to remove the resin, and the filtrate was concentrated to dryness under reduced pressure. Acetonitrile (4 mL) and purified water (8 mL) were added and lyophilized in a freeze dryer to obtain the title compound (230 mg).

[0646] LC-MS: m / z(ESI):1244.7[M+Na] +

[0647] Step 5: N-((4S,12S)-12-benzyl-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl-2,2-d2)-3,8,11,14 Synthesis of 1-(3-(2-(cyclopentanesulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propionamido)-3,6,9,12,15,18,21,24-octaoxaheptacosane-27-amide (b-L6-1)

[0648] Under a nitrogen atmosphere, N,N-dimethylformamide (2 mL) was added to a mixture of Intermediate L11-4 (230 mg), Intermediate L1-7 (87.64 mg), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (72.14 mg), 1-hydroxybenzotriazole (50.85 mg), and pyridine (44.65 mg). The resulting mixture was stirred at room temperature for 1 hour. After the reaction was complete as determined by LCMS, the reaction mixture was directly purified by HPLC (column: C18 Welch Xtimate C18 150*30mm*5um; mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 27%-47%, 22 min) to obtain the title compound (41.4 mg).

[0649] LC-MS:m / z(ESI):814.8[(M+2H) / 2] +

[0650] 1 H NMR (400MHz, DMSO-d6) δ = 9.24 (s, 1H), 8.70-8.64 (m, 1H), 8.60 (t, J = 6.6Hz, 1 H),8.29(t,J=5.9Hz,1H),8.17(t,J=5.6Hz,1H),8.11(d,J=8.0Hz,1H),8.07 (t,J=5.5Hz,1H),8.00(t,J=5.6Hz,1H),7.79(s,1H),7.52(s,1H),7.27-7.2 1(m,6H),7.16(s,1H),6.51(s,1H),5.43(s,4H),4.84-4.71(m,2H),4.68-4. 66(m,1H),4.54-4.46(m,1H),4.43-4.37(m,1H),4.28-4.19(m,1H),3.76-3. 66(m,11H),3.59(t,J=6.4Hz,3H),3.50-3.46(m,25H),3.26-3.18(m,5H),3. 05-3.03(m,1H),2.80-2.78(m,1H),2.42-2.40(m,5H),2.00-1.85(m,6H),1. 74-1.58(m,5H),0.99-0.96(m,1H),0.88(t,J=7.3Hz,3H),0.38-0.31(m,4H)

[0651] Example 33, (S)-N 4-(26-amino-3,6,9,12,15,18,21,24-octamethyl-2,5,8,11,14,17,20,23,26-nonaoxo-3,6,9,12,15,18,21,24-octaazahexacosyl)-N 1 -((4S,12S)-12-benzyl-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-8,10,11,13-tetrahydro-10H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl-2,2-d2)-3,8,11,14,17-pentaoxo-5-oxa-2,7,10,13,16-pentaazaoctadec-18-yl)-2-(3-(2-(tert-butylsulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propionamido)-N-((4S,12S)-12-benzyl-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-8,10,11,13-tetrahydro-10H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl-2,2-d2)-3,8,11,14,17-pentaoxo-5-oxa-2,7,10,13,16-pentaazaoctadec-18-yl)propionamido)-N-((4S,12S)-12-benzyl-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-8,10,11,13-tetrahydro-10H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1 4 -Methylsuccinamide (c-L12-1)

[0652] Step 1: Synthesis of (S)-5-allyloxycarbonyl-1-(9H-fluoren-9-yl)-8,11,14,17,20,23,26,29,32-nonamethyl-3,7,10,13,16,19,22,25,28,31-decaoxo-2-oxa-4,8,11,14,17,20,23,26,29,32-decaazatetratriacontane-34-carboxylic acid (Intermediate L12-1)

[0653] The compound was synthesized by peptide solid phase synthesis method, the steps are as follows:

[0654] 1) Under nitrogen, dichloromethane was added to a mixture of CTC resin (0.64 mmol / g, 12.5 g) and N-(((9H-fluoren-9-yl)methoxy)carbonyl)-N-methylglycine (2.50 g);

[0655] 2) Diisopropylethylamine (DIEA) (6.2 g) was added dropwise and the mixture was stirred for 2 h;

[0656] 3) Methanol (13 mL) was added and the mixture was stirred for 0.5 h;

[0657] 4) Filter and wash three times with DMF;

[0658] 5) Add 20% piperidine / DMF solution and react for 30 min;

[0659] 6) Filter and wash five times with DMF;

[0660] 7) Add the materials listed in the "Raw Materials" column in the table below and mix for 30 seconds. Then add the materials listed in the "Reagents" column in the table below. The reaction is carried out under nitrogen bubbling for 1 hour.

[0661] 8) Repeat steps 4-7 to complete the synthesis of the desired peptide.

[0662] Note:

[0663] After the solid phase was prepared, a 20% HFIP / DCM solution was added to the resulting mixture and stirred for 1.5 h. The mixture was then filtered, the filtrate collected, and concentrated. The resulting mixture was purified by preparative liquid chromatography (A: 0.075% TFA in H2O, B: acetonitrile) to afford the title compound (2.15 g).

[0664] MS m / z(ESI):1035.6[M+H] + .

[0665] Step 2: Synthesis of (S)-30-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-1-amino-3,6,9,12,15,18,21,24,27-nonamethyl-1,4,7,10,13,16,19,22,25,28-decaoxo-3,6,9,12,15,18,21,24,27-nonaazatriacontane-31-carboxylic acid allyl ester (Intermediate L12-2)

[0666] Intermediate L12-1 (1 g), ammonium chloride (155.09 mg), diisopropylethylamine (499.44 mg), HATU (728.95 mg) were dissolved in DMF (5 mL), and the atmosphere was replaced with nitrogen three times. The reaction was stirred at 25 ° C for 3 h. After the reaction was completed, the crude product was purified by reverse phase high performance liquid chromatography ( 80 g C 18 Column, mobile phase gradient 0-40% acetonitrile / water, flow rate 40 mL / min) to obtain the title compound (800 mg).

[0667] MS m / z(ESI):1034.6[M+H] + .

[0668] Step 3: Synthesis of (S)-30-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-1-amino-3,6,9,12,15,18,21,24,27-nonamethyl-1,4,7,10,13,16,19,22,25,28-decaoxo-3,6,9,12,15,18,21,24,27-nonaazatriacontane-31-carboxylic acid (Intermediate L12-3)

[0669] Intermediate L12-2 (800 mg), tetrakistriphenylphosphine palladium (201.84 mg), 1.3-dimethylbarbituric acid (108.71 mg) were dissolved in DMF (5 mL), and the atmosphere was replaced with nitrogen three times. The reaction was stirred at 25 ° C for 4 h. After the reaction was completed, the crude product was purified by reverse phase high performance liquid chromatography ( 80 g C18 column, mobile phase gradient 0-40% acetonitrile / water, flow rate 40 mL / min) to obtain the title compound (700 mg).

[0670] MS m / z(ESI):994.9[M+H] + .

[0671] Step 4: Synthesis of resin-supported (2S,10S,19S)-19-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-48-amino-10-benzyl-2-cyclopropyl-22,25,28,31,34,37,40,43,46-nonamethyl-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48-pentadecaoxo-3-oxa-5,8,11,14,17,22,25,28,31,34,37,40,43,46-tetraazatetraoctadecanoic acid (Intermediate L12-4)

[0672] Intermediate L12-3 (700 mg) was added to DMF (10 mL), followed by Intermediate L1-25 (resin plus substrate total weight 400 mg, content approximately 0.18 mmol), O-benzotriazole-tetramethyluronium hexafluorophosphate (336.58 mg), and diisopropylethylamine (205.62 mg). The reaction mixture was nitrogen-purged three times and shaken on a shaker at 25°C for 1 hour. After completion of the reaction, the resin was washed three times with methanol (10 mL) and then with dichloromethane (10 mL). The resin was then filtered to dryness and dried under vacuum to yield the title compound (420 mg).

[0673] MS m / z(ESI):1461.4[M+Na] + .

[0674] Step 5: Synthesis of resin-supported (2S,10S,19S)-19,48-diamino-10-benzyl-2-cyclopropyl-22,25,28,31,34,37,40,43,46-nonamethyl-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48-pentadecaoxo-3-oxa-5,8,11,14,17,22,25,28,31,34,37,40,43,46-tetraazatetraoctadecanoic acid (Intermediate L12-5)

[0675] Intermediate L12-4 (420 mg) was dissolved in DMF (5 mL) and piperidine (1.25 mL) was added. The reaction mixture was shaken at 25°C for 1 hour. After the reaction, the resin was washed with methanol (10 mL) and dichloromethane (10 mL) three times, followed by filtration to dryness and drying under vacuum to obtain the title compound (400 mg).

[0676] MS m / z(ESI):1217.4[M+H] + .

[0677] Step 6: Synthesis of resin-supported (2S,10S,19S)-48-amino-10-benzyl-19-(3-(2-(tert-butylsulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propionamido)-2-cyclopropyl-22,25,28,31,34,37,40,43,46-nonamethyl-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48-pentadecaoxo-3-oxa-5,8,11,14,17,22,25,28,31,34,37,40,43,46-tetraazaoctadecane-1-carboxylic acid (Intermediate L12-6)

[0678] Intermediate L12-5 (360 mg) and intermediate L4-1 (108.39 mg) were dissolved in DMF (10 mL). 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (114 mg) and pyridine (108 mg) were added. The reaction mixture was shaken at 25°C for 1 hour. After completion of the reaction, the resin was washed with methanol (10 mL) and dichloromethane (10 mL) three times, followed by filtration to dryness and drying under vacuum to obtain the title compound (390 mg). Step 7: Synthesis of (2S,10S,19S)-48-amino-10-benzyl-19-(3-(2-(tert-butylsulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propionamido)-2-cyclopropyl-22,25,28,31,34,37,40,43,46-nonamethyl-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48-pentadecaoxo-3-oxa-5,8,11,14,17,22,25,28,31,34,37,40,43,46-tetraazaoctadecane-1-carboxylic acid (Intermediate L12-7)

[0679] Intermediate L12-6 (390 mg) was added to a mixed solvent of dichloromethane (8 mL) and 1,1,1,3,3,3-hexafluoroisopropanol (2 mL) and shaken at 25°C for 0.5 h. After completion of the reaction, the reaction mixture was filtered to remove the resin, and the filtrate was concentrated to dryness under reduced pressure. The crude product was directly purified by HPLC (column: Boston Green ODS 150*30mm*5um; mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 10%-50%, 10 min) to obtain the title compound (25 mg).

[0680] Step 8: (S)-N 4 -(26-amino-3,6,9,12,15,18,21,24-octamethyl-2,5,8,11,14,17,20,23,26-nonaoxo-3,6,9,12,15,18,21,24-octaazahexacosyl)-N 1 -((4S,12S)-12-benzyl-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-8,10,11,13-tetrahydro-10H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl-2,2-d2)-3,8,11,14,17-pentaoxo-5-oxa-2,7,10,13,16-pentaazaoctadec-18-yl)-2-(3-(2-(tert-butylsulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propionamido)-N-((4S,12S)-12-benzyl-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-8,10,11,13-tetrahydro-10H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl-2,2-d2)-3,8,11,14,17-pentaoxo-5-oxa-2,7,10,13,16-pentaazaoctadec-18-yl)propionamido)-N-((4S,12S)-12-benzyl-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-8,10,11,13-tetrahydro-10H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1 4 Synthesis of -methylsuccinamide (c-L12-1)

[0681] Intermediate L7-7 (16 mg), intermediate L1-7 (4.4 mg), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (3.98 mg), pyridine (2.46 mg), and 1-hydroxybenzotriazole (2.81 mg) were dissolved in DMF (2 mL). The atmosphere was purged with nitrogen three times. The reaction mixture was stirred at 25°C under nitrogen for 2 h. After completion of the reaction, the reaction mixture was directly purified by HPLC (column: C18 100*10 mm; mobile phase: [A: water (ammonium bicarbonate), B: acetonitrile]; B%: 10%-38%, 16 min) to obtain the title compound (4.5 mg).

[0682] (Mobile phase A preparation: 30L pure water + 24g ammonium bicarbonate)

[0683] 1H NMR (400MHz, DMSO-d6) δ = 9.24 (s, 1H), 8.66 (d, J = 6.4Hz, 1H), 8.59 (s, 1H), 8. 27(s,2H),8.01(d,J=16.5Hz,2H),7.81(s,1H),7.52(s,1H),7.26(s,1H),7. 22(s,5H),7.16(s,1H),6.50(s,1H),5.44(d,J=9.9Hz,4H),4.85-4.71(m,3H ),4.70-4.63(m,1H),4.57(s,1H),4.49(s,1H),4.43-4.27(m,6H),4.23(d,J= 16.1Hz,3H),4.16(s,1H),4.13-3.99(m,5H),3.92(s,4H),3.86(d,J=13.0Hz ,2H),3.75-3.64(m,10H),3.59(s,2H),3.49(d,J=7.0Hz,1H),3.22(t,J=6.4 Hz,2H),2.99-2.84(m,14H),2.84-2.71(m,14H),1.92-1.79(m,2H),1.37(s, 9H),1.24(s,2H),0.96(d,J=7.7Hz,1H),0.91-0.84(m,3H),0.39-0.30(m,4H)

[0684] MS m / z(ESI):973.9[M / 2+H] + .

[0685] Example 34, N-((4S,12S)-12-benzyl-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolane-[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl-2,2-d2)-3 ,8,11,14,17-pentaoxo-5-oxa-2,7,10,13,16-pentaazaoctadec-18-yl)-6-(2-(cyclopentylsulfonyl)-5-oxo-8,8-di(2,5,8,11-tetraoxatridecan-13-yl)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)hexanamide (b-L13-1)

[0686] Step 1: Synthesis of 6-(2-cyclopentanesulfonyl-5-oxo-8,8-di(2,5,8,11-tetraoxatridecan-13-yl)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)hexanoic acid (Intermediate L13-1)

[0687] Compound 18 (800 mg) was dissolved in dichloromethane (8 mL) and trifluoroacetic acid (1.5 mL) was added. The reaction mixture was stirred at 25°C under nitrogen protection for 2 h. After the reaction was completed, the solvent was removed by concentration under reduced pressure. The residue was purified by rapid silica gel column chromatography ( 12g Flash silica gel column, gradient 0-30% tetrahydrofuran / petroleum ether, flow rate 50 mL / min) to give the title compound (280 mg).

[0688] MS m / z(ESI):776.6(M+H) + .

[0689] Step 2: Synthesis of resin-supported (2S,10S)-10-benzyl-23-(2-cyclopentanesulfonyl-5-oxo-8,8-di(2,5,8,11-tetraoxatridecan-13-yl)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)-2-cyclopropyl-6,9,12,15,18-pentaoxo-3-oxa-5,8,11,14,17-pentaazatricosanoic acid (Intermediate L13-2)

[0690] Intermediate L13-1 (280 mg) and intermediate L1-25 (440 mg) were dissolved in N,N-dimethylformamide (5 mL). O-Benzotriazole-tetramethyluronium hexafluorophosphate (183.22 mg) and N,N-diisopropylethylamine (139.91 mg) were added sequentially. The reaction solution was shaken at 25°C for 1 hour. After completion of the reaction, the resin was washed with methanol (10 mL) and dichloromethane (10 mL) three times, followed by filtration to dryness and drying under vacuum to obtain the title compound (470 mg).

[0691] MS m / z(ESI):1221.1(M+H) + .

[0692] Step 3: Synthesis of (2S,10S)-10-benzyl-23-(2-cyclopentanesulfonyl-5-oxo-8,8-di(2,5,8,11-tetraoxatridecan-13-yl)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)-2-cyclopropyl-6,9,12,15,18-pentaoxo-3-oxa-5,8,11,14,17-pentaazatricosanoic acid (Intermediate L13-3)

[0693] Intermediate L13-2 (450 mg) was dissolved in dichloromethane (8 mL) and 1,1,1,3,3,3-hexafluoroisopropanol (2 mL). The reaction mixture was shaken at 25°C for 0.5 h. After completion of the reaction, the mixture was filtered, and the filtrate was concentrated under reduced pressure to remove the solvent. The residue was purified by preparative HPLC (C18 column, 5 μm particle size, 30 mm diameter, 150 mm length; eluent: decreasingly polar mixtures of water (containing 0.225% formic acid) and acetonitrile; acetonitrile gradient ratio 25%-45% over 22 minutes) to yield the title compound (34 mg).

[0694] MS m / z(ESI):1221.4(M+H) + .

[0695] Step 4: N-((4S,12S)-12-benzyl-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl-2,2-d2)-3,8, Synthesis of 11,14,17-pentaoxo-5-oxa-2,7,10,13,16-pentaazaoctadec-18-yl)-6-(2-(cyclopentylsulfonyl)-5-oxo-8,8-di(2,5,8,11-tetraoxatridecan-13-yl)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)hexanamide (b-L13-1)

[0696] Intermediate L13-3 (34 mg) and intermediate L1-7 (12.38 mg) were dissolved in N,N-dimethylformamide (0.5 mL). 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (10.67 mg), 1-hydroxybenzotriazole (7.52 mg), and pyridine (6.61 mg) were added sequentially. The atmosphere was purged with nitrogen three times. The reaction mixture was stirred at 25°C under nitrogen for 1 hour. After completion of the reaction, the reaction mixture was directly purified by HPLC (C18 column, 5 μm particle size, 30 mm diameter, 150 mm length; eluent: decreasingly polar mixtures of water (containing 0.225% formic acid) and acetonitrile; acetonitrile gradient ratio 32%-52% over 22 minutes) to obtain the title compound (26.8 mg).

[0697] MS m / z(ESI):814.1[(M+2H) / 2] + .

[0698] 1H NMR (400MHz, DMSO-d6) δ = 9.24 (s, 1H), 8.71-8.64 (m, 1H), 8.62-8.57 (m, 1H), 8 .33-8.25(m,1H),8.12(d,J=7.9Hz,1H),8.09-8.04(m,1H),8.04-7.98(m,1H), 7.80(s,1H),7.52(s,1H),7.26-7.23(m,5H),7.19-7.12(m,1H),6.49(s,1H),5 .44(d,J=8.3Hz,4H),4.84-4.71(m,2H),4.70-4.63(m,1H),4.54-4.45(m,1H), 4.44-4.37(m,1H),4.31-4.21(m,1H),3.80-3.63(m,8H),3.63-3.57(m,1H),3 .53-3.36(m,30H),3.22(s,6H),3.09-3.00(m,1H),2.84-2.75(m,1H),2.13(d, J=7.5Hz,2H),2.11-1.93(m,8H),1.90(s,2H),1.68(m,4H),1.55(d,J=7.7Hz,4 H),1.33-1.25(m,2H),1.01-0.93(m,1H),0.90-0.85(m,3H),0.39-0.28(m,4H)

[0699] Example 35, N-((4S,12S,21S)-12-benzyl-21-(3-(2'-(cyclopentanesulfonyl)-5'-oxo-5'H-spiro[cyclopenta-1,8'-pyrido[4,3-d]pyrimidin]-6'(7'H)-yl)propionamido)-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3] Dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl-2,2-d2)-3,8,11,14,17,20-hexaoxo-5-oxa-2,7,10,13,16,19-hexaazapentacosan-25-yl)-2,5,8,11,14,17,20,23-octaoxahexacosan-26-amide (b-L14-1)

[0700] Step 1: Synthesis of 3-(2'-(cyclopentanesulfonyl)-5'-oxo-5'H-spiro[cyclopentane-1,8'-pyrido[4,3-d]pyrimidin]-6'(7'H)-yl)propanoic acid (Intermediate L14-1)

[0701] Compound 19 (630 mg) was dissolved in dichloromethane (3 mL), and trifluoroacetic acid (0.6 mL) was added. The reaction mixture was stirred at 25°C for 1 hour. After completion of the reaction, the mixture was concentrated under reduced pressure. The residue was slurried with petroleum ether (1 mL) and ethyl acetate (1 mL) and dried under vacuum to obtain the title compound (380 mg).

[0702] MS m / z(ESI):408.2[M+H] + .

[0703] Step 2: Synthesis of resin-supported (32S,41S,49S)-41-benzyl-32-(3-(2'-cyclopentanesulfonyl-5'-oxo-5'H-spiro[cyclopentane-1,8'-pyrido[4,3-d]pyrimidin]-6'(7'H)-yl)propionamido)-49-cyclopropyl-26,33,36,39,42,45-hexaoxo-2,5,8,11,14,17,20,23,48-nonaoxa-27,34,37,40,43,46-hexaazapentacontane-50-carboxylic acid (Intermediate L14-2)

[0704] Intermediate L14-1 was dissolved in N,N-dimethylformamide (3 mL), followed by the addition of Intermediate L2-3 (195.59 mg), O-benzotriazole-tetramethyluronium hexafluorophosphate (243.72 mg), and N,N-diisopropylethylamine (250.82 μL). The reaction mixture was shaken at 25°C for 1 hour. After completion of the reaction, the resin was washed three times with methanol (5 mL) and then with dichloromethane (5 mL). The resin was then filtered to dryness and dried under vacuum to yield the title compound (430 mg).

[0705] Step 3: Synthesis of (32S,41S,49S)-41-benzyl-32-(3-(2'-cyclopentanesulfonyl-5'-oxo-5'H-spiro[cyclopentane-1,8'-pyrido[4,3-d]pyrimidin]-6'(7'H)-yl)propionamido)-49-cyclopropyl-26,33,36,39,42,45-hexaoxo-2,5,8,11,14,17,20,23,48-nonaoxa-27,34,37,40,43,46-hexaazapentacontane-50-carboxylic acid (Intermediate L14-3)

[0706] Intermediate L14-2 (430 mg) was added to a mixed solvent of dichloromethane (4 mL) and 1,1,1,3,3,3-hexafluoroisopropanol (1 mL). The reaction mixture was shaken at 25°C for 1 hour. After completion of the reaction, the resin was removed by filtration, and the filtrate was concentrated under reduced pressure. The product was purified by HPLC (column: Welch Xtimate C18 150*30mm*5μm; mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 25%-45%, 15 min) to obtain the title compound (17 mg).

[0707] Step 4: N-((4S,12S,21S)-12-benzyl-21-(3-(2'-cyclopentanesulfonyl-5'-oxo-5'H-spiro[cyclopentane-1,8'-pyrido[4,3-d]pyrimidin]-6'(7'H)-yl)propionamido)-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxadiodeutetrahydro-10H-[1,3]dioxadiodeutetrahydro-10H-[1,3]dioxadiodeutetrahydro-10H-[1,3]dioxadiodeutetrahydro-10H-[1,3]dioxadiodeutetrahydro-10H-[1,3]dioxadiodeutetrahydro-10H-[1,3]dioxadiodeutetrahydro-10H-[1,3]dioxadiodeutetrahydro-10H-[1,3]dioxadiodeutetrahydro-10H-[1,3]dioxadiodeutetrahydro-10H-[1,8 ... Synthesis of Pentacyclo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl-2,2-d2)-3,8,11,14,17,20-hexaoxo-5-oxa-2,7,10,13,16,19-hexaazapentacosan-25-yl)-2,5,8,11,14,17,20,23-octaoxahexacosan-26-amide (b-L14-1)

[0708] Intermediate L14-3 (17 mg), intermediate L1-7 (5.76 mg), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (4.74 mg), pyridine (8.56 mg), and 1-hydroxybenzotriazole (3.34 mg) were dissolved in N,N-dimethylformamide (0.5 mL). The atmosphere was purged with nitrogen three times. The reaction mixture was stirred at 25°C under nitrogen for 2 hours. After completion of the reaction, the reaction mixture was directly purified by HPLC (column: Welch Xtimate C18 150*30mm*5um; mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 32%-52%, 22 min) to obtain the title compound (9.8 mg).

[0709] MS m / z(ESI):891.2[M / 2+H] + .

[0710] 1H NMR (400MHz, DMSO-d6) δ = 9.23 (s, 1H), 8.73-8.55 (m, 2H), 8.36-8.29 (m, 1H), 8.25-8.16 ( m,2H),8.13(d,J=7.9Hz,1H),7.99(t,J=5.1Hz,1H),7.81-7.74(m,2H),7.50(s,1H),7.25 (s,1H),7.23-7.19(m,4H),7.18-7.12(m,1H),6.49(s,1H),5.42(s,4H),4.84-4.71(m,2H ),4.66(m,J=6.7,9.8Hz,1H),4.55-4.45(m,1H),4.39(m,J=6.5,10.1Hz,1H),4.31-4.16( m,2H),3.70-3.66(m,7H),3.60-3.53(m,5H),3.51-3.47(m,25H),3.40(s,3H),3.23(s,3H ),3.08-2.90(m,4H),2.78(m,1H),2.27(t,J=6.5Hz,2H),2.07-1.91(m,7H),1.90-1.82(m ,2H),1.76(s,6H),1.71-1.57(m,6H),1.54-1.43(m,1H),1.36-1.30(m,2H),1.28-1.23(m ,2H),1.00-0.92(m,1H),0.87(t,J=7.3Hz,3H),0.38-0.34(m,2H),0.31(d,J=4.5Hz,2H).

[0711] Example 36, (S)-N-((4S,12S)-12-benzyl-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolane-[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl-2,2-d2)-3,8,11,14,17-pentahydro 1-(2-(2-(2-(2-cyclopentanesulfonyl-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin]-6(5H)-yl)propionamido)-14-(2,5,8,11-tetraoxatridecan-13-yl)-2,5,8,11-tetraoxatridecan-14-yl)-2,5,8,11-tetraoxatridecan-20-amide (b-L15-1)

[0712] Step 1: Synthesis of 2,5,8,11-tetraoxatridecan-13-aldehyde (Intermediate L15-1)

[0713] To a solution of oxalyl chloride (914 mg) in dichloromethane (25 mL) was slowly added dropwise dimethyl sulfoxide (1.02 mL) at -70°C under a nitrogen atmosphere. The reaction mixture was stirred at -70°C for 10 minutes. A solution of 2,5,8,11-tetraoxatridecan-13-ol (1.00 g) in dichloromethane (5 mL) was then added dropwise at -70°C. The reaction mixture was stirred at -70°C for 10 minutes. Triethylamine (4.02 mL) was then added dropwise at -70°C. The reaction mixture was allowed to warm to room temperature and stirred at 20°C for 0.5 h. After completion of the reaction, a solution of the title compound in dichloromethane (30 mL) was obtained, which was filtered and used directly in the next reaction.

[0714] Step 2: Synthesis of (S)-19-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-14-(2,5,8,11-tetraoxatridecan-13-yl)-2,5,8,11-tetraoxa-14-azaeicosane-20-carboxylic acid tert-butyl ester (Intermediate L15-2)

[0715] At room temperature and nitrogen atmosphere, sodium triacetoxyborohydride (4.07 g) was added to a solution of Fmoc-lysine tert-butyl ester hydrochloride (407.58 mg) in ethanol (20 mL). A solution of the intermediate L15-1 obtained in the previous step in dichloromethane (30 mL) was added dropwise. The resulting mixture was stirred at 20°C for 3 h. After completion of the reaction, the solvent was removed under reduced pressure, and the residue was purified by flash silica gel column chromatography ( 20g Flash silica gel column, gradient 0-15% methanol / dichloromethane, flow rate 60 mL / min) to give the title compound (1.00 g).

[0716] LC-MS: m / z(ESI):805.6[M+H] + .

[0717] Step 3: Synthesis of (S)-19-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-14-(2,5,8,11-tetraoxatridecan-13-yl)-2,5,8,11-tetraoxa-14-azaeicosane-20-carboxylic acid (Intermediate L15-3)

[0718] Under nitrogen atmosphere, intermediate L15-2 (1.00 g) was dissolved in dichloromethane (5 mL), trifluoroacetic acid (5 mL) was added at room temperature, and the reaction solution was reacted at 20°C for 2 h. After the reaction was completed, the solvent was removed under reduced pressure, and the residue was purified by flash silica gel column chromatography ( 12g Flash silica gel column, gradient 0-15% methanol / dichloromethane, flow rate 60 mL / min) to give the title compound (330 mg).

[0719] LC-MS: m / z(ESI):749.4[M+H] + .

[0720] Step 4: Synthesis of resin-supported (19S,28S,36S)-19-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-28-benzyl-36-cyclopropyl-20,23,26,29,32-pentaoxo-14-(2,5,8,11-tetraoxatridecan-13-yl)-2,5,8,11,35-pentaoxa-14,21,24,27,30,33-hexaazaheptatriacontane-37-carboxylic acid (Intermediate L15-4)

[0721] Intermediate L15-3 (301.44 mg) and intermediate L1-25 (400 mg) were dissolved in anhydrous N,N-dimethylformamide (8 mL). O-Benzotriazole-tetramethyluronium hexafluorophosphate (88.86 mg) and N,N-diisopropylethylamine (113.09 mg) were added sequentially. The reaction mixture was shaken at 25°C for 1 hour. After completion of the reaction, the resin was washed with methanol (20 mL) and dichloromethane (20 mL) three times, followed by filtration to dryness and drying under vacuum to obtain the title compound (500 mg).

[0722] LC-MS: m / z(ESI):1194.6[M+H] + .

[0723] Step 5: Synthesis of resin-supported (19S,28S,36S)-19-amino-28-benzyl-36-cyclopropyl-20,23,26,29,32-pentaoxo-14-(2,5,8,11-tetraoxatridecan-13-yl)-2,5,8,11,35-pentaoxa-14,21,24,27,30,33-hexaazaheptatriacontane-37-carboxylic acid (Intermediate L15-5)

[0724] Intermediate L15-4 (0.55 g) was dissolved in anhydrous N,N-dimethylformamide (2.5 mL), and piperidine (0.5 mL) was added. The reaction mixture was shaken at 25°C for 1 hour. After completion of the reaction, the resin was washed three times with methanol (20 mL) and then with dichloromethane (20 mL). The resin was then filtered to dryness and dried under vacuum to obtain the title compound (440 mg).

[0725] LC-MS: m / z(ESI):972.6[M+H] + .

[0726] Step 6: Synthesis of resin-supported (19S,28S,36S)-28-benzyl-19-(3-(2-cyclopentanesulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin]-6(5H)-yl)propionamido)-36-cyclopropyl-20,23,26,29,32-pentaoxo-14-(2,5,8,11-tetraoxatridecan-13-yl)-2,5,8,11,35-pentaoxa-14,21,24,27,30,33-hexaazaheptatriacontane-37-carboxylic acid (Intermediate L15-6)

[0727] Intermediate L15-5 (400 mg) and intermediate L3-1 (100.74 mg) were dissolved in anhydrous N,N-dimethylformamide (8 mL). O-Benzotriazole-tetramethyluronium hexafluorophosphate (48.25 mg) and N,N-diisopropylethylamine (122.80 mg) were added sequentially. The reaction mixture was shaken at 25°C for 1 hour. After the reaction, the resin was washed with methanol (20 mL) and dichloromethane (20 mL) three times. The resin was then filtered to dryness and dried under vacuum to obtain the title compound (458 mg).

[0728] LC-MS: m / z(ESI):1307.5[M+H] + .

[0729] Step 7: Synthesis of (19S,28S,36S)-28-benzyl-19-(3-(2-cyclopentanesulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin]-6(5H)-yl)propionamido)-36-cyclopropyl-20,23,26,29,32-pentaoxo-14-(2,5,8,11-tetraoxatridecan-13-yl)-2,5,8,11,35-pentaoxa-14,21,24,27,30,33-hexaazaheptatriacontane-37-carboxylic acid (Intermediate L15-7)

[0730] Intermediate L15-6 (458.00 mg) was dissolved in anhydrous dichloromethane (4 mL), and 1,1,1,3,3,3-hexafluoroisopropanol (1 mL) was added. The reaction mixture was stirred at 25°C for 1 hour. After completion of the reaction, the mixture was filtered and washed three times with dichloromethane (10 mL). The filtrate was concentrated under reduced pressure and purified by preparative HPLC (C18 column, 5 μm particle size, 30 mm diameter, 150 mm length; eluent: decreasingly polar mixtures of water (containing 0.225% formic acid) and acetonitrile; acetonitrile gradient ratio 20%-37%, elution time 19 minutes) to obtain the title compound (25 mg).

[0731] LC-MS: m / z(ESI):1307.5[M+H]+ .

[0732] Step 8: (S)-N-((4S,12S)-12-benzyl-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl-2,2-d2)-3,8,11,14,17-pentaoxo- Synthesis of 5-oxa-2,7,10,13,16-pentaazaoctadec-18-yl)-19-(3-(2-cyclopentanesulfonyl-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin]-6(5H)-yl)propionamido)-14-(2,5,8,11-tetraoxatridecan-13-yl)-2,5,8,11-tetraoxa-14-azaeicosane-20-amide (b-L15-1)

[0733] Intermediate L15-7 (20.00 mg) and intermediate L1-7 (14.25 mg) were dissolved in anhydrous N,N-dimethylformamide (0.8 mL). 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (11.73 mg), 1-hydroxybenzotriazole (8.27 mg), and pyridine (7.26 mg) were added sequentially. The atmosphere was purged with nitrogen three times. The reaction mixture was stirred at 25°C under nitrogen for 1 hour. After completion of the reaction, the reaction mixture was directly purified by HPLC (C18 column, 5 μm particle size, 30 mm diameter, 150 mm length; eluent: a decreasingly polar mixture of water (containing 0.225% formic acid) and acetonitrile; acetonitrile gradient ratio 28%-38.5% over 15 minutes) to obtain the title compound (7.8 mg).

[0734] LC-MS:m / z(ESI):857.4[(M+2H) / 2] + .

[0735] 1H NMR (400MHz, DMSO-d6)δ=9.29-9.17(m,1H),8.73-8.59(m,2H),8.34(s,2 H),8.30-8.18(m,2H),8.17-8.10(m,1H),8.04-7.96(m,1H),7.82-7.75(m ,1H),7.54-7.46(m,1H),7.29-7.18(m,5H),7.18-7.12(m,1H),6.68-6.4 1(m,1H),5.46-5.40(m,3H),4.87-4.71(m,2H),4.70-4.62(m,1H),4.56-4 .34(m,2H),4.29-4.14(m,2H),3.78-3.60(m,11H),3.53-3.44(m,32H),3 .23-3.21(m,6H),3.07-3.00(m,1H),2.84-2.74(m,1H),2.58-2.53(m,4H) ,2.40-2.32(m,2H),1.80(m,6H),1.75-1.56(m,5H),1.53-1.39(m,1H),1 .37-1.13(m,4H),1.00-0.93(m,1H),0.91-0.83(m,3H),0.41-0.26(m,4H)

[0736] Example 37, (S)-N 4 -(26-amino-3,6,9,12,15,18,21,24-octamethyl-2,5,8,11,14,17,20,23,26-nonaoxo-3,6,9,12,15,18,21,24-octaazahexacosan-1-yl)-N 1 -((4S,12S)-12-benzyl-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxadiodeuterino[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl-2,2-d2)-3,8,11,14,17-pentaoxo-5-oxa-2,7,10,13,16-pentaazaoctadec-18-yl)-2-(3-(2-(cyclopentanesulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propionamido)-N 4 -Methylsuccinamide (b-L12-1)

[0737] Step 1: Synthesis of resin-supported (2S,10S,19S)-48-amino-10-benzyl-19-(3-(2-(cyclopentanesulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propionamido)-2-cyclopropyl-22,25,28,31,34,37,40,43,46-nonamethyl-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48-pentadecaoxo-3-oxa-5,8,11,14,17,22,25,28,31,34,37,40,43,46-tetraazatetraoctadecane-1-carboxylic acid (Intermediate L16-1)

[0738] Intermediate L12-5 (535 mg) and intermediate L3-1 (187.41 mg) were dissolved in N,N-dimethylformamide (10 mL). O-benzotriazole-tetramethyluronium hexafluorophosphate (270 mg) and diisopropylethylamine (309 μL) were added. The reaction mixture was shaken at 25°C for 1 hour. After completion of the reaction, the resin was washed with methanol (10 mL) and dichloromethane (10 mL) three times, followed by filtration to dryness and drying under vacuum to obtain the title compound (550 mg).

[0739] Step 2: Synthesis of (2S,10S,19S)-48-amino-10-benzyl-19-(3-(2-(cyclopentanesulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propionamido)-2-cyclopropyl-22,25,28,31,34,37,40,43,46-nonamethyl-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48-pentadecaoxo-3-oxa-5,8,11,14,17,22,25,28,31,34,37,40,43,46-tetraazaoctadecane-1-carboxylic acid (Intermediate L16-2)

[0740] Intermediate L16-1 (550 mg) was added to a mixed solvent of dichloromethane (8 mL) and 1,1,1,3,3,3-hexafluoroisopropanol (2 mL) and shaken at 25°C for 0.5 hr. After completion of the reaction, the reaction mixture was filtered to remove the resin, and the filtrate was concentrated to dryness under reduced pressure. The residue was directly purified by HPLC (column: Welch Xtimate C18 150*30mm*5um; mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 18%-38%, 20 min) to obtain the title compound (35 mg).

[0741] Step 3: (S)-N 4-(26-amino-3,6,9,12,15,18,21,24-octamethyl-2,5,8,11,14,17,20,23,26-nonaoxo-3,6,9,12,15,18,21,24-octaazahexacosan-1-yl)-N 1 -((4S,12S)-12-benzyl-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxadiodeuterino[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl-2,2-d2)-3,8,11,14,17-pentaoxo-5-oxa-2,7,10,13,16-pentaazaoctadec-18-yl)-2-(3-(2-(cyclopentanesulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propionamido)-N 4 Synthesis of -methylsuccinamide (b-L12-1)

[0742] Intermediate L16-2 (35 mg), intermediate L1-7 (9.54 mg), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (8.64 mg), pyridine (5.35 mg), and 1-hydroxybenzotriazole (6.09 mg) were dissolved in N,N-dimethylformamide (1 mL). The atmosphere was purged with nitrogen three times. The reaction mixture was stirred at 25°C under nitrogen for 2 hours. After completion of the reaction, the reaction mixture was directly purified by HPLC (column: Welch Xtimate C18 150*30mm*5um; mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 25%-45%, 20 min) to obtain the title compound (8.2 mg).

[0743] 1H NMR (400MHz, DMSO-d6) δ = 9.22 (s, 1H), 8.72-8.64 (m, 1H), 8.59 (d, J = 4.9Hz, 1H), 8.35-8.21 (m, 2H), 8.09 -7.91(m,2H),7.80(s,1H),7.52(s,1H),7.35-7.19(m,5H),7.18-7.15(m,1H),7.11-7.04(m,1H),6.53- 6.49(m,1H),5.44(d,J=9.8Hz,3H),4.83-4.30(m,9H),4.28-3.82(m,13H),3.78-3.48(m,18H),3.08-2.64(m,30H),2.04-1 .80(m,6H),1.73-1.56(m,4H),1.53-1.39(m,1H),1.24(s,3H),1.01-0.92(m,1H),0.87(t,J=7.2Hz,3H),0.43-0.26(m,4H)

[0744] MS m / z(ESI):979.6[(M+2H) / 2] + .

[0745] Example 38, N-((4S,12S,21S)-12-benzyl-21-(3-(2'-(cyclopentanesulfonyl)-5'-oxo-2,3,5,6-tetrahydro-5'H-spiro[pyran-4,8'-pyrido[4,3-d]pyrimidin]-6'(7'H)-yl)propionamido)-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H -[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl-2,2-d2)-3,8,11,14,17,20-hexaoxo-5-oxa-2,7,10,13,16,19-hexaazapentacosan-25-yl)-2,5,8,11,14,17,20,23-octaoxahexacosan-26-amide (b-L17-1)

[0746] Step 1: Synthesis of 3-(2'-(cyclopentanesulfonyl)-5'-oxo-2,3,5,6-tetrahydro-5'H-spiro[pyran-4,8'-pyrido[4,3-d]pyrimidin]-6'(7'H)-yl)propanoic acid (Intermediate L17-1)

[0747] Compound 20 (680 mg) was dissolved in dichloromethane (10 mL), and trifluoroacetic acid (0.68 mL) was added to the reaction solution. The reaction solution was stirred at 25°C for 1 hour. After the reaction was completed, the solvent was directly removed by concentration under reduced pressure. The residue was purified by flash silica gel column chromatography ( 12g Flash silica gel column, gradient 0-53% tetrahydrofuran / petroleum ether, flow rate 40 mL / min) to obtain the title compound (200 mg)

[0748] MS m / z(ESI):423.8[M+H] +

[0749] Step 2: Synthesis of resin-supported (32S,41S,49S)-41-benzyl-32-(3-(2'-(cyclopentanesulfonyl)-5'-oxo-2,3,5,6-tetrahydro-5'H-spiro[pyran-4,8'-pyrido[4,3-d]pyrimidin]-6'(7'H)-yl)propionamido)-49-cyclopropyl-26,33,36,39,42,45-hexaoxo-2,5,8,11,14,17,20,23,48-nonaoxa-27,34,37,40,43,46-hexaazapentacontane-50-carboxylic acid (Intermediate L17-2)

[0750] Intermediate L2-3 (400 mg) and intermediate L17-1 (106.13 mg) were dissolved in N,N-dimethylformamide (10 mL). O-Benzotriazole-tetramethyluronium hexafluorophosphate (159.06 mg) and N,N-diisopropylethylamine (121.46 mg) were added sequentially, and the reaction mixture was shaken at 25°C for 1 hour. After completion of the reaction, the resin was washed with methanol (10 mL) and dichloromethane (10 mL) three times, followed by filtration to dryness and drying under vacuum to obtain the title compound (420 mg).

[0751] MS m / z(ESI):1391.5[M+H] + .

[0752] Step 3: Synthesis of (32S,41S,49S)-41-benzyl-32-(3-(2'-(cyclopentanesulfonyl)-5'-oxo-2,3,5,6-tetrahydro-5'H-spiro[pyran-4,8'-pyrido[4,3-d]pyrimidin]-6'(7'H)-yl)propionamido)-49-cyclopropyl-26,33,36,39,42,45-hexaoxo-2,5,8,11,14,17,20,23,48-nonaoxa-27,34,37,40,43,46-hexaazapentacontane-50-carboxylic acid (Intermediate L17-3)

[0753] Intermediate L17-2 (400 mg) was dissolved in dichloromethane (8 mL) and 1,1,1,3,3,3-hexafluoroisopropanol (2 mL). The reaction mixture was shaken at 25°C for 0.25 h. After completion, the reaction mixture was filtered and concentrated under reduced pressure to remove the solvent. The residue was purified by preparative HPLC (C18 column, 5 μm particle size, 30 mm diameter, 150 mm length; eluent: decreasingly polar mixtures of water (containing 0.225% formic acid) and acetonitrile; acetonitrile gradient ratio 20%-40% over 22 minutes) to yield the title compound (15 mg).

[0754] MS m / z(ESI):1413.4[M+Na] + .

[0755] Step 4: N-((4S,12S,21S)-12-benzyl-21-(3-(2'-(cyclopentanesulfonyl)-5'-oxo-2,3,5,6-tetrahydro-5'H-spiro[pyran-4,8'-pyrido[4,3-d]pyrimidin]-6'(7'H)-yl)propionamido)-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1 Synthesis of (b-L17-1)-3,8,11,14,17,20-hexaoxo-5-oxa-2,7,10,13,16,19-hexaazapentacosan-25-yl)-2,5,8,11,14,17,20,23-octaoxahexacosan-26-amide

[0756] Intermediate L17-3 (15 mg) and intermediate L1-7 (4.79 mg) were dissolved in N,N-dimethylformamide (0.5 mL). 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (4.13 mg), 1-hydroxybenzotriazole (2.91 mg), and pyridine (2.56 mg) were added sequentially. The atmosphere was purged with nitrogen three times. The reaction mixture was stirred at 25°C under nitrogen for 1 hour. After completion of the reaction, the reaction mixture was directly purified by HPLC (C18 column, 5 μm particle size, 30 mm diameter, 150 mm length; eluent: decreasingly polar mixtures of water (containing 0.225% formic acid) and acetonitrile; acetonitrile gradient from 28% to 48% over 22 minutes) to yield the title compound (9 mg).

[0757] MS m / z(ESI):899.1[(M+2H) / 2] + .

[0758] 1 H NMR (400MHz, DMSO-d6) δ = 9.28 (s, 1H), 8.66 (s, 1H), 8.62-8.56 (m, 1H), 8.30 (s, 1H), 8.21 (s,1H),8.17-8.13(m,1H),8.13-8.08(m,1H),8.01-7.95(m,1H),7.82-7.75(m,2H),7.53 -7.50(m,1H),7.26(s,1H),7.24-7.21(m,4H),7.19-7.13(m,1H),6.48(s,1H),5.44(d,J =8.9Hz,4H),4.86-4.73(m,2H),4.70-4.64(m,1H),4.52-4.48(m,1H),4.44-4.36(m,1H), 4.29(s,1H),4.24-4.17(m,1H),3.83-3.80(m,3H),3.74-3.67(m,10H),3.58-3.55(m,3H ),3.52-3.48(m,28H),3.43-3.42(m,3H),3.24(s,3H),3.06-2.97(m,3H),2.83-2.75(m,1 H),2.31-2.26(m,2H),2.08-1.94(m,6H),1.90-1.81(m,2H),1.70-1.60(m,7H),1.53-1.4 5(m,1H),1.38-1.31(m,2H),1.24(s,2H),0.96(s,1H),0.88(t,J=7.4Hz,3H),0.36(s,4H)

[0759] Example 39, N-((4S,12S)-12-benzyl-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolane-[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl-2,2-d2)-3,8,11,14,17-pentaoxo -5-oxa-2,7,10,13,16-pentaazaoctadec-18-yl)-6-(2'-(cyclopentanesulfonyl)-1-(2,5,8,11,14,17,20,23-octaoxa-26-oxohexacosa-26-yl)-5'-oxo-5'H-spiro[piperidin-4,8'-pyrido[4,3-d]pyrimidin]-6'(7'H)-yl)hexanamide (b-L18-1)

[0760] Step 1: Synthesis of benzyl 6'-(6-tert-butoxy-6-oxohexyl)-2'-(cyclopentylthio)-5'-oxo-6',7'-dihydro-5'H-spiro[piperido-4,8'-pyrido[4,3-d]pyrimidine]-1-carboxylate (Intermediate L18-1)

[0761] Under a nitrogen atmosphere at 0°C, intermediate 18-3 (3 g) was dissolved in N,N-dimethylformamide (40 mL). NaH (1.72 g, 60% active ingredient) was added. The reaction was stirred at 0°C for 15 min, and then benzyl bis(2-iodoethyl)carbamate (9.85 g) was added. The reaction mixture was stirred at 0°C under nitrogen for 1 hour. After completion of the reaction, the mixture was poured into 50 mL of ice water and extracted with ethyl acetate (20 mL x 2). The combined organic phases were dried over anhydrous sodium sulfate, concentrated under reduced pressure, and separated by column chromatography (tetrahydrofuran / petroleum ether, 0-18%) to obtain the title compound (2.7 g).

[0762] LC-MS: m / z(ESI):623.2[M+H] + .

[0763] Step 2: Synthesis of 6-(2'-(cyclopentylthio)-5'-oxo-5'H-spiro[piperido-4,8'-pyrido[4,3-d]pyrimidin]-6'(7'H)-yl)hexanoic acid (Intermediate L18-2)

[0764] Intermediate L18-1 (2.7 g) was dissolved in acetonitrile (5 mL), and trimethylsilyl iodide (1.23 mL) was added. The reaction mixture was stirred at 25°C for 0.5 h. The reaction mixture was purified by column chromatography (column: Spherical C18 20-35 μm 100A 40 g; mobile phase: [A: water, B: acetonitrile]; B%: 10%-30%, 60 min) to obtain the title compound (1.4 g).

[0765] LC-MS: m / z(ESI):433.3[M+H] + .

[0766] Step 3: Synthesis of 6-(2'-(cyclopentylthio)-1-(2,5,8,11,14,17,20,23-octaoxa-26-oxohexacosa-26-yl)-5'-oxo-5'H-spiro[piperido-4,8'-pyrido[4,3-d]pyrimidin]-6'(7'H)-yl)hexanoic acid (Intermediate L18-3)

[0767] Intermediate L18-2 (500 mg) and 2,5-dioxopyrrolidin-1-yl 2,5,8,11,14,17,20,23-octaoxahexacosa-26-oate (647.86 mg) were dissolved in anhydrous N,N-dimethylformamide (2 mL). N,N-diisopropylethylamine (302 μL) was added, and the reaction mixture was stirred at 25°C for 1 hour. The reaction mixture was purified by column chromatography (column: Spherical C18 20-35 μm 100A 40 g; mobile phase: [A: water, B: acetonitrile]; B%: 10%-30%, 60 min) to obtain the title compound (900 mg).

[0768] LC-MS: m / z(ESI):827.6[M+H] + .

[0769] Step 4: Synthesis of 6-(2'-(cyclopentanesulfonyl)-1-(2,5,8,11,14,17,20,23-octaoxa-26-oxohexacosa-26-yl)-5'-oxo-5'H-spiro[piperido-4,8'-pyrido[4,3-d]pyrimidin]-6'(7'H)-yl)hexanoic acid (Intermediate L18-4)

[0770] Intermediate L18-3 (0.9 g) was dissolved in anhydrous dichloromethane (10 mL) at 0°C, and m-chloroperbenzoic acid (375.58 mg) was added. The reaction was stirred at 25°C for 1 hour. After completion of the reaction, the mixture was concentrated under reduced pressure at low temperature, and the residue was purified by column chromatography (column: Spherical C18 20-35 μm 100A 40 g; mobile phase: [A: water, B: acetonitrile]; B%: 10%-24%, 60 min) to obtain the title compound (472 mg).

[0771] LC-MS: m / z(ESI):859.5[M+H] + .

[0772] Step 5: Synthesis of resin-supported (2S,10S)-10-benzyl-23-(2'-(cyclopentanesulfonyl)-1-(2,5,8,11,14,17,20,23-octaoxa-26-oxohexacosa-26-yl)-5'-oxo-5'H-spiro[piperidin-4,8'-pyrido[4,3-d]pyrimidin]-6'(7'H)-yl)-2-cyclopropyl-6,9,12,15,18-pentaoxo-3-oxa-5,8,11,14,17-pentaazatricosane-1-carboxylic acid (Intermediate L18-5)

[0773] Under a nitrogen atmosphere, N,N-dimethylformamide (6 mL) and N,N-diisopropylethylamine (213.38 μL) were added to a mixture of intermediate L1-25 (308 mg), intermediate L18-4 (466 mg), and benzotriazole-N,N,N,N-tetramethyluronium hexafluorophosphate (309.72 mg). The resulting mixture was shaken on a shaker at 20°C for 1 hr. After the reaction, the resin was washed with methanol (20 mL) and dichloromethane (20 mL) three times, followed by filtration to dryness and drying under vacuum to obtain the title compound (350 mg).

[0774] LC-MS:m / z(ESI):653.0[(M+2H) / 2] +

[0775] Step 6: Synthesis of (2S,10S)-10-benzyl-23-(2'-(cyclopentanesulfonyl)-1-(2,5,8,11,14,17,20,23-octaoxa-26-oxohexacosa-26-yl)-5'-oxo-5'H-spiro[piperidin-4,8'-pyrido[4,3-d]pyrimidin]-6'(7'H)-yl)-2-cyclopropyl-6,9,12,15,18-pentaoxo-3-oxa-5,8,11,14,17-pentaazatricosane-1-carboxylic acid (Intermediate L18-6)

[0776] Under a nitrogen atmosphere, intermediate L18-5 (350 mg) was dissolved in dichloromethane (10 mL) and 1,1,1,3,3,3-hexafluoroisopropanol (2.5 mL). The resulting mixture was shaken at 25°C for 0.5 hours. After completion of the reaction, the resin was removed by filtration, and the filtrate was concentrated to dryness under reduced pressure. The product was then purified by HPLC (column: C18 Welch Xtimate C18 150*30mm*5um; mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 22%-52%, 11 min) to yield the title compound (10 mg).

[0777] LC-MS:m / z(ESI):653.1[(M+2H) / 2] +

[0778] Step 7: N-((4S,12S)-12-benzyl-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl-2,2-d2)-3,8,11,14,17-pentaoxo-5-(4-hydroxy-1-(4-hydroxy-1-(4-hydroxy-1-(4-hydroxy-1-(4-hydroxy-1-(4-hydroxy-1-(4-hydroxy-1-(4-hydroxy-1-(4-hydroxy-1-(4-hydroxy-1-(4-hydroxy-1-(4-hydroxy-1-(4-hydroxy-1-(4-hydroxy-1-(4-hydroxy-1-(4-hydroxy-1-(4-hydroxy-1-(4-hydroxy-1- Synthesis of b-L18-1

[0779] Under a nitrogen atmosphere, N,N-dimethylformamide (2 mL) was added to a mixture of Intermediate L18-6 (10 mg), Intermediate L1-7 (3.25 mg), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.94 mg), 1-hydroxybenzotriazole (2.07 mg), and pyridine (1.85 μL). The resulting mixture was stirred at room temperature for 1 hour. After the reaction was complete as determined by LCMS, the reaction mixture was directly purified by HPLC (column: C18 Welch Xtimate C18 150*30mm*5μm; mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 28%-48%, 20 min) to obtain the title compound (7.3 mg).

[0780] LC-MS:m / z(ESI):855.9[(M+2H) / 2] +

[0781] 1H NMR (400MHz, DMSO-d6) δ = 9.28 (s, 1H), 8.67 (s, 1H), 8.60 (s, 1H), 8.29 (s, 1 H),8.15-8.06(m,2H),8.04-7.99(m,1H),7.80(s,1H),7.52(s,1H),7.28-7 .19(m,5H),7.16(s,1H),6.50(s,1H),5.44(d,J=9.9Hz,4H),4.77(s,2H), 4.71-4.63(m,1H),4.49(s,1H),4.39(d,J=7.0Hz,1H),4.26(t,J=6.9Hz,1H ),3.95(s,1H),3.77(s,3H),3.71-3.61(m,6H),3.54-3.45(m,32H),3.44- 3.40(m,6H),3.04(d,J=10.1Hz,1H),2.84-2.75(m,1H),2.65-2.56(m,2H), 2.14(t,J=7.0Hz,2H),2.03-1.83(m,8H),1.73-1.50(m,10H),1.26(d,J=1 1.8Hz,2H),0.95(d,J=5.4Hz,1H),0.87(t,J=7.2Hz,3H),0.40-0.28(m,4H)

[0782] Example 40, (S)-N 4 -(26-amino-3,6,9,12,15,18,21,24-octamethyl-2,5,8,11,14,17,20,23,26-nonaoxo-3,6,9,12,15,18,21,24-octaazahexacosan-1-yl)-N 1 -((4S,12S)-12-benzyl-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxadiodeuteropentano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl-2,2-d2)- 3,8,11,14,17-pentaoxo-5-oxa-2,7,10,13,16-pentaazaoctadec-18-yl)-2-(3-(2'-(cyclopentanesulfonyl)-5'-oxo-2,3,5,6-tetrahydro-5'H-spiro[pyran-4,8'-pyrido[4,3-d]pyrimidin]-6'(7'H)-yl)propanamido)-N 4 -Methylsuccinamide (b-L19-1)

[0783] Step 1: Resin-supported (2S,10S,19S)-48-amino-10-benzyl-19-(3-(2'-(cyclopentanesulfonyl)-5'-oxo-2,3,5,6-tetrahydro-5'H-spiro[pyran-4,8'-pyrido[4,3-d]pyrimidin]-6'(7'H)-yl)propionamido)-2-cyclopropyl-22,25,28,31,34, Synthesis of 37,40,43,46-nonamethyl-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48-pentadeca-3-oxa-5,8,11,14,17,22,25,28,31,34,37,40,43,46-tetraazaoctadecane-1-carboxylic acid (Intermediate L19-1)

[0784] Intermediate L12-5 (880 mg) and intermediate L17-1 (393.14 mg) were dissolved in N,N-dimethylformamide (15 mL). O-benzotriazole-tetramethyluronium hexafluorophosphate (453.65 mg) and N,N-diisopropylethylamine (313.82 μL) were added. The reaction mixture was shaken at 25°C for 1 hour. After completion of the reaction, the resin was washed with methanol (10 mL) and dichloromethane (10 mL) three times, followed by filtration to dryness and drying under vacuum to obtain the title compound (920 mg).

[0785] Step 2: (2S,10S,19S)-48-amino-10-benzyl-19-(3-(2-(cyclopentanesulfonyl)-5'-oxo-2,3,5,6-tetrahydro-5'H-spiro[pyran-4,8'-pyrido[4,3-d]pyrimidin]-6'(7'H)-yl)propionamido)-2-cyclopropyl-22,25,28,31,34,37, Synthesis of 40,43,46-nonamethyl-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48-pentadeca-3-oxa-5,8,11,14,17,22,25,28,31,34,37,40,43,46-tetraazaoctadecane-1-carboxylic acid (Intermediate L19-2)

[0786] Intermediate L19-1 (920 mg) was added to a mixed solvent of dichloromethane (8 mL) and 1,1,1,3,3,3-hexafluoroisopropanol (2 mL) and shaken at 25°C for 0.5 hr. After completion of the reaction, the reaction mixture was filtered to remove the resin, and the filtrate was concentrated to dryness under reduced pressure. The residue was directly purified by HPLC (column: Welch Xtimate C18 150*30mm*5um; mobile phase: [Phase A: water (0.225% formic acid), Phase B: acetonitrile]; B%: 18%-38%, 20 min) to obtain the title compound (65 mg).

[0787] Step 3: (S)-N 4 -(26-amino-3,6,9,12,15,18,21,24-octamethyl-2,5,8,11,14,17,20,23,26-nonaoxo-3,6,9,12,15,18,21,24-octaazahexacosane)-N 1 -((4S,12S)-12-benzyl-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl-2,2-d2)-3,8,11,14,17-pentaoxo-5-oxa-2,7,10,13,16-pentaazaoctadec-18-yl)-2-(3-(2'-(cyclopentanesulfonyl)-5'-oxo-2,3,5,6-tetrahydro-5'H-spiro[pyran-4,8'-pyrido[4,3-d]pyrimidin]-6'(7'H)-yl)propionamido)-N-((4S,12S)-12-benzyl-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl-2,2-d2)-3,8,11,14,17-pentaoxo-5-oxa-2,7,10,13,16-pentaazaoctadec-18-yl)propionamido)-N-((4S,12S)-12-benzyl-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolano[4,5- 4 Synthesis of -methylsuccinamide (b-L19-1)

[0788] Intermediate L19-2 (65 mg), intermediate L1-7 (22.10 mg), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (15.36 mg), pyridine (9.51 mg), and 1-hydroxybenzotriazole (10.82 mg) were dissolved in N,N-dimethylformamide (3 mL). The atmosphere was purged with nitrogen three times. The reaction mixture was stirred at 25°C under nitrogen for 2 hours. After completion of the reaction, the reaction mixture was directly purified by HPLC (column: Welch Xtimate C18 150*30mm*5um; mobile phase: [Phase A: water (0.225% formic acid), Phase B: acetonitrile]; B%: 21%-51%, 11 min) to obtain the title compound (35 mg).

[0789] 1H NMR (400MHz, DMSO-d6) δ = 9.28 (s, 1H), 8.69-8.55 (m, 2H), 8.30-8.21 (m, 2H), 8.01 (m, 2H), 7.79 (s, 1H), 7.52 (s, 1H), 7.39-7.27 (m, 1H), 7. 26-7.21(m,5H),7.15(s,1H),7.11-7.00(m,1H),6.49(s,1H),5.43(d,J=5.8Hz,4H),4.86-4.70(m,2H),4.70-4.63(m,1H),4.62-4.53(m,1 H),4.52-4.44(m,1H),4.40-4.17(m,10H),4.12-3.89(m,9H),3.86-3.76(m,5H),3.75-3.65(m,9H),3.63-3.52(m,2H),3.51-3.43(m,2H) ,3.00-2.68(m,32H),2.07-1.92(m,6H),1.91-1.80(m,2H),1.71-1.59(m,6H),1.01-0.92(m,1H),0.88(t,J=7.3Hz,3H),0.38-0.27(m,4H)

[0790] MS m / z(ESI):1014.8[(M+2) / 2] + .

[0791] Example 41, ((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamic acid 4-((30S,33S,36S)-1-amino-30-(3-(2'-(cyclopentanesulfonyl)-5'-oxo-2,3,5,6-tetrahydro-5'H-spiro[pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamic acid pyrimidine]-6'(7'H)-yl)propionamido)-3,3-isopropyl-3,6,9,12,15,18,21,24,27,36-decamethyl-1,4,7,10,13,16,19,22,25,28,31,34-dodecaoxo-3,6,9,12,15,18,21,24,27,32,35-undecaazaheptatriacontane-37-carboxamido)benzyl ester (compound b-L22-3)

[0792] Step 1: Synthesis of tert-butyl ((S)-3-methyl-1-(((S)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxobutanoate (Intermediate L22-2)

[0793] Intermediate L22-1 (1.9 g) and di(4-nitrophenyl) carbonate (1.76 g) were dissolved in DMF (20 mL), and DIEA (2.5 g) was added under nitrogen atmosphere. The reaction solution was stirred at 25 ° C for 3 h. The reaction solution was directly concentrated to remove the solvent, and the crude product was purified by rapid silica gel column ( 20g The title compound (2.5 g) was purified by flash silica gel column, eluent 0-45% tetrahydrofuran / petroleum ether, gradient @ 65 mL / min).

[0794] MS m / z(ESI):559.2[M+H] +

[0795] Step 2: Synthesis of 4-((S)-2-((S)-2-((tert-Butoxycarbonyl)amino)-3-methylbutanamido)propionamido)benzyl ((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (Intermediate L22-3)

[0796] Intermediate L22-2 (1 g) and isotecan mesylate (999.20 mg) were dissolved in DMF (17 mL) and pyridine (3 mL). HOBt (266.10 mg) and DIEA (277.65 mg, 374.19 uL) were added under a nitrogen atmosphere. The reaction mixture was stirred at 25°C for 3 h. The reaction mixture was then directly purified by HPLC to obtain the title compound (1.1 g).

[0797] MS m / z(ESI):855.2[M+H] + .

[0798] Step 3: Synthesis of 4-((S)-2-((S)-2-amino-3-methylbutanamido)propionamido)benzyl ((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (Intermediate L22-4)

[0799] To a solution of intermediate L22-3 (1.1 g) in dichloromethane (20 mL) was added trifluoroacetic acid (5 mL) at room temperature, and the reaction mixture was stirred at 25° C. for 1 h. The reaction mixture was then concentrated and dried in vacuo to obtain the title compound (0.9 g).

[0800] MS m / z(ESI):755.5[M+H] +

[0801] Step 4: ((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate 4-((30S,33S,36S)-30-((((9H-fluoren-9-yl)methoxy)carbonyl)-1H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate Synthesis of 1-amino-3,3-isopropyl-3,6,9,12,15,18,21,24,27,36-decamethyl-1,4,7,10,13,16,19,22,25,28,31,34-dodecaoxo-3,6,9,12,15,18,21,24,27,32,35-undecaazaheptatriacontane-37-amino)benzyl ester (Intermediate L22-5)

[0802] Intermediate L22-4 (135 mg) and intermediate L12-3 (97.79 mg) were dissolved in DMF (2 mL). HBTU (74.61 mg) and DIEA (38.14 mg) were added under a nitrogen atmosphere. The reaction mixture was stirred at 25°C for 3 h. The reaction mixture was then directly purified on a C18 reverse-phase column (acetonitrile:water = 0-50%) to obtain the title compound (120 mg).

[0803] MS m / z(ESI):1732.1[M+H] +

[0804] Step 5: ((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate 4-((30S,33S,36S)-1,30-diamino-3-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate Synthesis of 3-isopropyl-3,6,9,12,15,18,21,24,27,36-decamethyl-1,4,7,10,13,16,19,22,25,28,31,34-dodecaoxo-3,6,9,12,15,18,21,24,27,32,35-undecaazaheptatriacontane-37-amino)benzyl ester (Intermediate L22-6)

[0805] Intermediate L22-5 (60 mg) was dissolved in DMF (0.5 mL), piperidine (0.1 mL) was added under nitrogen atmosphere, and the reaction solution was stirred at 25° C. for 1 h. The reaction solution was then directly purified by C18 reverse phase column (acetonitrile:water = 0-50%) to purify the title compound (50 mg).

[0806] MS m / z(ESI):1508.8[M+H] +

[0807] Step 6: ((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamic acid 4-((30S,33S,36S)-1-amino-30-(3-(2'-(cyclopentanesulfonyl)-5'-oxo-2,3,5,6-tetrahydro-5'H-spiro[pyrano[ Synthesis of 4,8'-pyrido[4,3-d]pyrimidinyl]-6'(7'H)-yl)propionamido)-33-isopropyl-3,6,9,12,15,18,21,24,27,36-decamethyl-1,4,7,10,13,16,19,22,25,28,31,34-dodecaoxo-3,6,9,12,15,18,21,24,27,32,35-undecaazaheptatriacontane-37-carboxamido)benzyl ester (Compound b-L22-3)

[0808] To a solution of Intermediate L22-6 (40 mg) and Intermediate L17-1 (13.47 mg) in DMF (1 mL) were added HBTU (20.11 mg) and DIEA (10.28 mg, 13.86 μL) at room temperature. The reaction mixture was stirred at 25°C for 1 h. The reaction mixture was then directly purified by HPLC (column: C18 150 × 30 mm × 5 μm; mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 26%-56%, 20 min) to obtain the title compound (18.6 mg).

[0809] MS m / z(ESI):1915.5[M+H] + .

[0810] 1H NMR (400MHz, DMSO-d6) δ=9.68-9.42(m,1H),9.35-8.93(m,1H),8.21-8.17(m,2H),8.06-8.02(m,2H),7.78(d,J=10 .8Hz,2H),7.66-7.62(m,3H),7.37(d,J=8.7Hz,2H),7.32(s,1H),5.45(s,2H),5.29(s,3H),5.08(s,2H),4.79-4.63 (m,1H),4.41-4.16(m,10H),4.13-3.90(m,10H),3.84(s,4H),3.75-3.62(m,5H),3.01-2.72(m,32H),2.38(s,3H), 2.26-2.14(m,4H),2.09-1.85(m,8H),1.72-1.60(m,5H),1.550-1.46(m,2H),1.38-1.30(m,3H),0.91-0.81(m,9H).

[0811] Example 42, ((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamic acid 4-((42S,45S,48S)-1-amino-42-(3-(2'-(cyclopentylsulfonyl)-5'-oxo-2,3,5,6-tetrahydro-5'H-spiro[pyrano-4,8'-pyrido[4,3-d] pyrimidinyl]-6'(7'H)-yl)propionylamino)-45-isopropyl-3,6,9,12,15,18,21,24,27,30,33,36,39,48-tetradecamethyl-1,4,7,10,13,16,19,22,25,28,31,34,37,40,43,46-hexadecanoyl-3,6,9,12,15,18,21,24,27,30,33,36,39,44,47-pentadecaazanonatetradecane-49-amido)benzyl ester (compound b-L23-3)

[0812] Step 1: Synthesis of (S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(((S)-1-(((S)-1-((4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)aminocarbonyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-4-oxobutanoic acid allyl ester (Intermediate L23-2)

[0813] Intermediate L22-4 (300 mg) and compound L23-1 (86.44 mg) were dissolved in DMF (4 mL), and HBTU (110.99 mg) and DIEA (84.76 mg) were added. The reaction mixture was stirred at 25°C under a nitrogen atmosphere for 1 h. The mixture was then directly purified by reverse phase chromatography (column: C18 (12 g); mobile phase: [A: water, B: acetonitrile]; B%: 0%-100%, 60 mL / min) to obtain the title compound (180 mg).

[0814] MS m / z(ESI):1132.6[M+H] + .

[0815] Step 2: Synthesis of (S)-3-amino-4-(((S)-1-(((S)-1-((4-((((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)aminocarbonyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-4-oxobutanoic acid allyl ester (Intermediate L23-3)

[0816] Intermediate L23-2 (170 mg) was dissolved in DMF (1.5 mL) and piperidine (0.3 mL) and stirred at 25°C under nitrogen for 1 h. The product was then directly purified by reverse phase chromatography (column: C18 (40 g); mobile phase: [A: water, B: acetonitrile]; B%: 0%-40%, 60 mL / min) to afford the title compound (100 mg).

[0817] MS m / z(ESI):910.4[M+H] + .

[0818] Step 3: (S)-3-(3-(2'-(cyclopentanesulfonyl)-5'-oxo-2,3,5,6-tetrahydro-5'H-spiro[pyran-4,8'-pyrido[4,3-d]pyrimidin]-6'(7'H)-yl)propionamido)-4-(((S)-1-(((S)-1-((4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13 Synthesis of allyl 4-oxobutyrate (Intermediate L23-4)

[0819] Intermediate L23-3 (30 mg) and intermediate L17-1 (15.36 mg) were dissolved in DMF (1 mL), and HBTU (25.01 mg) and DIEA (12.78 mg) were added. The reaction mixture was stirred at 25°C under nitrogen for 1 h. The mixture was then directly purified by reverse phase chromatography (column: C18 (12 g); mobile phase: [A: water, B: acetonitrile]; B%: 0%-100%, 40 mL / min) to obtain the title compound (15 mg).

[0820] MS m / z(ESI):1315.5[M+H] + .

[0821] Step 4: (S)-3-(3-(2'-(cyclopentanesulfonyl)-5'-oxo-2,3,5,6-tetrahydro-5'H-spiro[pyran-4,8'-pyrido[4,3-d]pyrimidin]-6'(7'H)-yl)propionamido)-4-(((S)-1-(((S)-1-((4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10, Synthesis of 1,3-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-4-oxobutanoic acid (Intermediate L23-5)

[0822] Intermediate L23-4 (14 mg) was dissolved in DMF (0.5 mL), and tetrakistriphenylphosphine palladium (3.69 mg) and 1,3-barbituric acid (2.49 mg) were added. The reaction was stirred at 25°C under nitrogen for 1 h. The mixture was then directly purified by reverse phase chromatography (column: C18 (12 g); mobile phase: [A: water, B: acetonitrile]; B%: 0%-90%, 40 mL / min) to obtain the title compound (13 mg).

[0823] MS m / z(ESI):1275.2[M+H] + .

[0824] Step 5: Synthesis of N-(N-(((9H-fluoren-9-yl)methoxy)carbonyl)-N-methylglycyl)-N-methylglycine tert-butyl ester (Intermediate L23-6)

[0825] Fmoc-sarcosine (50 g) was dissolved in dichloromethane (500 mL). HATU (66.65 g) and DIEA (83.92 mL) were added to the reaction mixture. The resulting mixture was stirred at 25°C for 1 hour. N-methylglycine tert-butyl ester hydrochloride (29.17 g) was then added to the reaction mixture. The reaction mixture was stirred at 25°C for 2 hours. The mixture was then diluted with 200 mL of water and extracted with dichloromethane (100 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the title compound (115 g).

[0826] LC-MS: m / z(ESI):439.2[M+H] +

[0827] Step 6: Synthesis of N-(N-(((9H-fluoren-9-yl)methoxy)carbonyl)-N-methylglycyl)-N-methylglycine (Intermediate L23-7)

[0828] Intermediate L23-6 (110 g) was dissolved in dioxane hydrochloride (400 mL) and the reaction mixture was stirred at 25°C for 16 h. After completion of the reaction, the mixture was concentrated under reduced pressure and separated by column chromatography (methanol / dichloromethane, 0-9%) to obtain the title compound (40 g).

[0829] LC-MS: m / z(ESI):383.2[M+H] +

[0830] Step 7: Synthesis of 1-(9H-fluoren-9-yl)-4,7,10,13,16,19,22,25,28,31,34,37,40-tridecamethyl-3,6,9,12,15,18,21,24,27,30,33,36,39-trideca-2-oxa-4,7,10,13,16,19,22,25,28,31,34,37,40-trideca-tetradecanoic-42-oic acid (Intermediate L23-8)

[0831] The compound was synthesized by peptide solid phase synthesis method, the steps are as follows:

[0832] 9) Under nitrogen, dichloromethane was added to a mixture of CTC resin (1.17 mmol / g, 15 g) and N-(((9H-fluoren-9-yl)methoxy)carbonyl)-N-methylglycine (5.5 g);

[0833] 10) Diisopropylethylamine (DIEA) (40 mL) was added dropwise and the mixture was stirred for 16 h;

[0834] 11) The resin was filtered and washed three times with methanol (100 mL) and DCM (100 mL);

[0835] 12) Add 20% piperidine / DMF solution and react for 1 h;

[0836] 13) The resin was filtered and washed three times with methanol (100 mL) and DCM (100 mL);

[0837] 14) Add intermediate L23-7 (2.0 eq.) based on the weight of the resin, followed by HBTU (3 eq.) and DIEA (6.0 eq.). The reaction was continued under nitrogen bubbling for 1 h.

[0838] 15) Repeat steps 3-6 five times.

[0839] After the solid phase was prepared, 20% HFIP / DCM solution (12.5 mL) was added to the resulting mixture and stirred for 1 h. The mixture was then filtered and the filtrate was collected and concentrated to obtain the title compound (150 mg).

[0840] MS m / z(ESI):1164.8[M+H] + .

[0841] Step 8: Synthesis of (9H-fluoren-9-yl)methyl (38-amino-3,6,9,12,15,18,21,24,27,30,33,36-dodecamethyl-2,5,8,11,14,17,20,23,26,29,32,35,38-tridecaoxo-3,6,9,12,15,18,21,24,27,30,33,36-dodecaazatriadecyl)(methyl)carbamate (Intermediate L23-9)

[0842] Intermediate L23-8 (130 mg) was dissolved in DMF (2 mL), and ammonium chloride (17.92 mg), HATU (84.25 mg), and DIEA (11.97 μL) were added sequentially. The reaction mixture was stirred at 25°C for 1 hour. After completion of the reaction, the product was purified using a reverse-phase column (Spherical C18 20-35 μm 100A 20 g; mobile phase: [A: water, B: acetonitrile]; B%: 10%-30%, 60 min) to obtain the title compound (80 mg).

[0843] MS m / z(ESI):1163.6[M+H] + .

[0844] Step 9: Synthesis of N-(2-amino-2-oxoethyl)-N-methyl-2-(N,5,8,11,14,17,20,23,26,29,32-undecanmethyl-4,7,10,13,16,19,22,25,28,31-decaoxo-2,5,8,11,14,17,20,23,26,29,32-undecanazatetratriacontane-34-carboxamido)acetamide (Intermediate L23-10)

[0845] Intermediate L23-9 (70 mg) was dissolved in DMF (0.5 mL) and diethylamine (30.99 μL) was added. The reaction mixture was stirred at 25°C for 2 h. After completion of the reaction, the product was purified using a reverse-phase column (Spherical C18 20-35 μm 100A 20 g; mobile phase: [A: water, B: acetonitrile]; B%: 10%-30% over 60 min) to obtain the title compound (32 mg).

[0846] LC-MS: m / z(ESI):941.3[M+H] +

[0847] Step 10: ((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate 4-((42S,45S,48S)-1-amino-42-(3-(2'-(cyclopentylsulfonyl)-5'-oxo-2,3,5,6-tetrahydro-5'H-spiro[pyrano-4,8'-pyrido[4,3-d]pyrimidine Synthesis of 1,4,7,10,13,16,19,22,25,28,31,34,37,40,43,46-hexadecanoyl-3,6,9,12,15,18,21,24,27,30,33,36,39,44,47-pentadecaazanonadecan-49-amido)benzyl ester (compound b-L23-3)

[0848] Intermediate L23-5 (13 mg) and intermediate L23-10 (9.59 mg) were dissolved in DMF (0.5 mL), and HBTU (7.73 mg) and DIEA (3.95 mg) were added. The reaction mixture was stirred at 25°C under nitrogen for 1 hour. After completion of the reaction, the reaction mixture was filtered and purified by preparative HPLC (Welch Xtimate C18 column, 5 μm silica, 30 mm diameter, 150 mm length; eluent: decreasingly polar mixtures of water (containing 0.225% formic acid) and acetonitrile; acetonitrile gradient from 26% to 56% over 11 minutes) to obtain the title compound (2.7 mg).

[0849] MS m / z(ESI):1099.9[(M+2H) / 2] +

[0850] 1H NMR (400MHz, DMSO-d6) δ = 9.67-9.42 (m, 1H), 9.29 (s, 1H), 8.46-8.24 (m, 1H), 8.16-8.02 (m, 2H), 7.78 (d, J = 11.0Hz, 1H), 7. 69-7.61(m,2H),7.37(d,J=7.9Hz,2H),7.31(s,1H),6.53(s,1H),5.45(s,2H),5.29(s,3H),5.08(s,2H),4.78-4.64(m,1H ),4.42-4.19(m,14H),4.12-3.89(m,14H),3.84(s,5H),3.74-3.62(m,4H),3.18-3.05(m,2H),3.00-2.65(m,45H),2.38(s ,3H),2.25-2.14(m,2H),2.11-1.76(m,9H),1.72-1.61(m,5H),1.54-1.40(m,1H),1.37-1.29(m,3H),0.93-0.80(m,10H).

[0851] Example 43, ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)(methyl)carbamic acid 4-((30S,33S,36S)-1-amino-30-(3-(2' -(cyclopentanesulfonyl)-5'-oxo-2,3,5,6-tetrahydro-5'H-spiro[pyran-4,8'-pyrido[4,3-d]pyrimidin]-6'(7'H)-yl)propionamido)-33-isopropyl-3,6,9,12,15,18,21,24,27-nonamethyl-1,4,7,10,13,16,19,22,25,28,31,34-dodecaoxo-36-(3-ureidopropyl)-3,6,9,12,15,18,21,24,27,32,35-undecaazaheptatriacontane-37-amido)benzyl ester (Compound b-L26-4)

[0852] Step 1: Synthesis of pentafluorophenyl 3-(2'-(cyclopentanesulfonyl)-5'-oxo-2,3,5,6-tetrahydro-5'H-spiro[pyran-4,8'-pyrido[4,3-d]pyrimidin]-6'(7'H)-yl)propanoate (Intermediate L26-1)

[0853] Intermediate L17-1 (0.5 g) and pentafluorophenol (325.98 mg) were dissolved in DMF (5 mL). EDCI (425.68 mg) was added under nitrogen, and the reaction mixture was stirred at 25°C for 1 h. The reaction mixture was then directly purified on a C18 reverse-phase column (100% acetonitrile) to obtain the title compound (600 mg).

[0854] MS m / z(ESI):590.3[M+H] +

[0855] Step 2: Synthesis of (S)-1,30-diamino-3,6,9,12,15,18,21,24,27-nonamethyl-1,4,7,10,13,16,19,22,25,28-decaoxo-3,6,9,12,15,18,21,24,27-nonaazahexadecane-31-oic acid (Intermediate L26-2)

[0856] Intermediate L12-3 (1 g) was dissolved in tetrahydrofuran (10 mL) and water (5 mL), wet palladium on carbon (244.36 mg) was added, and the reaction mixture was stirred at 25°C for 16 h under a hydrogen atmosphere. The reaction mixture was then directly filtered and concentrated to obtain the title compound (0.7 g).

[0857] MS m / z(ESI):772.3[M+H] + .

[0858] Step 3: Synthesis of (S)-1-amino-30-(3-(2'-(cyclopentanesulfonyl)-5'-oxo-2,3,5,6-tetrahydro-5'H-spiro[pyrano-4,8'-pyrido[4,3-d]pyrimidin]-6'(7'H)-yl)propionamido)-3,6,9,12,15,18,21,24,27-nonamethyl-1,4,7,10,13,16,19,22,25,28-decaoxo-3,6,9,12,15,18,21,24,27-nonaazatridecane-31-oic acid (Intermediate L26-3)

[0859] Intermediate L26-2 (300 mg) and intermediate L26-1 (206.23 mg) were dissolved in DMF (3 mL). DIEA (150.71 mg) was added under a nitrogen atmosphere, and the reaction mixture was stirred at 25°C for 1 hour. The reaction mixture was then directly purified by HPLC (column: C18 150 × 30 mm × 5 μm; mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 13%-33%, 10 min) to obtain the title compound (100 mg).

[0860] MS m / z(ESI):1177.5[M+H] +

[0861] Step 4: ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamic acid 4-((30S,33S,36S)-1-amino-30-(3-(2'-( Synthesis of cyclopentanesulfonyl)-5'-oxo-2,3,5,6-tetrahydro-5'H-spiro[pyran-4,8'-pyrido[4,3-d]pyrimidin]-6'(7'H)-yl)propionylamino)-33-isopropyl-3,6,9,12,15,18,21,24,27-nonamethyl-1,4,7,10,13,16,19,22,25,28,31,34-dodecaoxo-36-(3-ureidopropyl)-3,6,9,12,15,18,21,24,27,32,35-undecaazaheptatriacontanoyl-37-amido)benzyl ester (compound b-L26-4)

[0862] Intermediate L26-3 (20 mg) and compound L26-4 (19.08 mg) were dissolved in DMF (0.5 mL) at room temperature. n-Butylphosphoric trianhydride (T4P) (24.48 mg, 50% active ingredient) and DIEA (8.78 mg, 11.84 μL) were added under a nitrogen atmosphere. The reaction mixture was stirred at 25°C for 1 h. The reaction mixture was then directly purified by HPLC (column: C18 150×40 mm; mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 20%-60%, 9 min) to obtain the title compound (11 mg).

[0863] MS m / z(ESI):1142.6[(M+2H) / 2] +

[0864] 1H NMR (400MHz, DMSO-d6) δ=9.80-9.51(m,1H),9.32-9.23(m,1H),8.40-8.27(m,1H),8.18-7.95(m,2H),7.89(d,J=9.0Hz,1H ),7.62(s,2H),7.33-7.23(m,6H),7.17(s,2H),5.97(s,1H),5.40(s,3H),5.15-4.91(m,2H),4.79-4.57(m,2H),4.49(d,J =5.9Hz,2H),4.37-4.19(m,10H),4.13-3.66(m,22H),3.61-3.54(m,1H),3.25-3.17(m,9H),3.12(s,2H),3.02-2.71(m,34 H),2.31-2.23(m,1H),2.16-1.90(m,11H),1.84-1.57(m,12H),1.56-1.20(m,8H),1.06-0.97(m,6H),0.91-0.72(m,26H).

[0865] Example 44, (2-(2-chloro-4-(3-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)ureido)phenethoxy)ethyl)(methyl)carbamic acid 4-((30S,33S,36S)-1-amino-30-(3-(2'-(cyclopentanesulfonyl)-5'-oxo-2,3,5,6-tetrahydro-5'H-spiro[pyran-4,8'-pyrido[4,3-d]pyrimidine]-6-yl)-1-(2-(2-chloro-4-(3-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)ureido)phenethoxy)ethyl)(methyl)carbamic acid '(7'H)-yl)propionylamino)-33-isopropyl-3,6,9,12,15,18,21,24,27-nonamethyl-1,4,7,10,13,16,19,22,25,28,31,34-dodecaoxo-36-(3-ureidopropyl)-3,6,9,12,15,18,21,24,27,32,35-undecaazaheptatriacontane-37-amido)benzyl ester (Compound b-L27-5)

[0866] Step 1: Synthesis of 4-((S)-2-((S)-2-((tert-Butoxycarbonyl)amino)-3-methylbutyrylamino)-5-ureidopentanamido)benzyl (2-(2-chloro-4-(3-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)ureido)phenethoxy)ethyl)(methyl)carbamate (Intermediate L27-3)

[0867] Compound L27-1 (100 mg) and compound L27-2 (90 mg, synthesized according to the method reported in patent WO2021198965) were dissolved in DMF (1 mL), DIEA (60.08 mg) was added under nitrogen atmosphere, and the reaction solution was stirred at 25 ° C for 1 h. The reaction solution was then diluted with ethyl acetate (30 mL) and washed with water (30 mL × 3), and the combined organic phases were dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by rapid silica gel column ( 20g The title compound (100 mg) was purified by flash silica gel column, eluent: 0-100% tetrahydrofuran / petroleum ether, gradient @ 40 mL / min).

[0868] MS m / z(ESI):1033.3[M+H] + .

[0869] Step 2: Synthesis of 4-((S)-2-((S)-2-amino-3-methylbutyrylamino)-5-ureidopentanamido)benzyl (2-(2-chloro-4-(3-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)ureido)phenethoxy)ethyl)(methyl)carbamate (Intermediate L27-4)

[0870] To a solution of intermediate L27-3 (100 mg) in dichloromethane (1 mL) was added trifluoroacetic acid (0.2 mL) at 0°C, and the reaction mixture was stirred at 25°C for 1 h. The reaction mixture was then concentrated, dried under vacuum, and purified by HPLC (column: C18 150 × 30 mm; mobile phase: [A: water (0.00025% hydrochloric acid), B: acetonitrile]; B%: 12%-52%, 9 min) to afford the title compound (13 mg).

[0871] MS m / z(ESI):933.6[M+H] +

[0872] Step 3: (2-(2-chloro-4-(3-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)ureido)phenethoxy)ethyl)(methyl)carbamic acid 4-((30S,33S,36S)-1-amino-30-(3-(2'-(cyclopentanesulfonyl)-5'-oxo-2,3,5,6-tetrahydro-5'H-spiro[pyran-4,8'-pyrido[4,3-d]pyrimidine]-6'(7 Synthesis of 3,3-isopropyl-3,6,9,12,15,18,21,24,27-nonamethyl-1,4,7,10,13,16,19,22,25,28,31,34-dodecaoxo-36-(3-ureidopropyl)-3,6,9,12,15,18,21,24,27,32,35-undecaazaheptatriacontane-37-amido)benzyl ester (compound b-L27-5)

[0873] To a solution of intermediate L27-4 (11 mg) and compound L26-3 (13.87 mg) in DMF (0.5 mL) were added n-butylphosphoric trianhydride (12.73 mg, 50% content) and DIEA (4.57 mg, 6.16 μL) at room temperature. The reaction was stirred at 25°C for 1 h. The reaction solution was then directly purified by HPLC (column: C18 150×30 mm; mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 22%-62%, 9 min) to obtain the title compound (6.5 mg).

[0874] MS m / z(ESI):1046.9[(M+2H) / 2] +

[0875] 1H NMR (400MHz, DMSO-d6) δ = 10.99 (s, 1H), 9.79-9.52 (m, 1H), 9.29 (s, 1H), 8.79 (s, 1 H),8.41-8.21(m,1H),8.10-7.90(m,2H),7.73-7.58(m,4H),7.52(s,1H),7.44(d ,J=8.1Hz,1H),7.30(s,2H),7.23-7.05(m,3H),6.81(t,J=5.6Hz,1H),5.98(d,J= 2.3Hz,1H),5.42(s,2H),5.13-5.09(m,1H),4.98(s,2H),4.81-4.65(m,1H),4.41( d,J=8.0Hz,3H),4.37-4.19(m,10H),4.14-3.99(m,6H),3.97-3.86(m,4H),3.85- 3.74(m,6H),3.73-3.65(m,3H),3.59-3.45(m,8H),3.02-2.89(m,12H),2.89-2.7 3(m,22H),2.64-2.57(m,2H),2.41-2.37(m,1H),2.09-1.91(m,8H),1.84-1.75(m ,1H),1.70-1.61(m,6H),1.48-1.44(m,3H),1.39-1.31(m,2H),0.89-0.78(m,8H).

[0876] Example 45. Preparation or Source of Drug-Linker Controls 1 to 5

[0877] The preparation of drug-linker control 1 refers to patent document CN111065621A,

[0878] Drug-linker control 2 was prepared by the method reported in Example 22 and patent document CN111065621A.

[0879] Drug-linker control 3 was purchased from MCE (catalog number: HY-15575).

[0880] The preparation of drug-linker control 4 refers to patent document WO2021198965A1,

[0881] Drug-linker control 5 was purchased from MCE (catalog number: HY-147270).

[0882] Test Example 1: Physical and Chemical Property Test of Antibody Conjugates

[0883] 1.1 Antibodies:

[0884] 1.1.1 Construction and production of anti-human HER2 monoclonal antibodies

[0885] The sequences of the anti-human HER2 monoclonal antibody are shown in Table 1 below. The heavy and light chain nucleotide sequences were cloned into the pTT5 vector (purchased from Ubest Biosciences), and plasmids were prepared according to established standard molecular biology methods. The expression vector and transfection reagent PEI (Polysciences, Catalog No. 24765-1) were added to OPTI-MEM (Gibco, Catalog No. 11058021), mixed thoroughly, and allowed to stand for 15 minutes. The cells were then added to Expi293F cells (Thermofisher, Catalog No. A14527) and incubated in a shaking incubator at 37°C with 5% CO2 and 120 rpm. On the second day of transfection, OPM-293 ProFeed (Shanghai Aopuma, Catalog No. F081918-001) and 6 g / L glucose (Sigma, Catalog No. G7528) were added. On the sixth day of transfection, the cell supernatant was collected.

[0886] Table 1 Anti-human HER2 antibody Trastuzumab amino acid sequence information and CDR analysis (according to Kabat classification)

[0887] 1.1.2 Construction and production of anti-human EGFR and cMet bispecific antibodies

[0888] Referring to the method of Example 1.1.1, the nucleic acid sequences encoding the antibody VH and VL were used to construct plasmids (sequence source WO2024002938A1, see Table 2), and transfected into Expi293F cells to obtain expression supernatant.

[0889] Table 2 Sequence information of anti-human EGFR-cMet bispecific antibody EGFR-cMet-Ab

[0890] 1.1.3 Construction and production of anti-human CDH6 antibodies

[0891] The anti-human CDH6 antibody CDH6-Ab-1 was produced by immunizing mice. The immunogens were human CDH6-hFc protein and HEK293T cells overexpressing human CDH6. Hybridoma cells were prepared from splenic lymphocytes of mice with high antibody titers in serum. Positive hybridoma clones were screened using conventional methods in the art, such as ELISA and FACS. Antibodies were further prepared using serum-free cell culture, purified, and sequenced to obtain the murine anti-human CDH6 antibody and its variable region sequence. By comparing the IMGT (http: / / imgt.cines.fr) human antibody heavy and light chain variable region germline gene database and MOE (Molecular Operating Environment) software, we selected heavy and light chain variable region germline genes with high homology to the mouse antibody as templates. The mouse antibody CDRs were transplanted into the corresponding human templates, forming a variable region sequence with the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Based on this, back mutations were performed as needed to maintain the original affinity and / or hotspot mutations were performed to eliminate the risk of molecular modification. The final optimized humanized antibody CDH6-Ab-1 corresponding VH and VL sequences and CDR sequences classified according to Kabat format are shown in Table 3.

[0892] Table 3 Sequence information of anti-human CDH6 antibody CDH6-Ab-1 and its CDR sequence (according to Kabat classification)

[0893] Referring to the method of Example 1.1.1, the nucleic acid sequences encoding the antibody VH and VL were used to construct plasmids, and the plasmids were transfected into Expi293F cells to obtain expression supernatant.

[0894] 1.2 Antibody Purification

[0895] Purify the above-mentioned antibodies from cell culture supernatant using Protein A affinity chromatography (Cytiva, Cat. No. 17549802). Wash the Protein A affinity column with 0.5M NaOH for 3-5 column volumes, followed by 3-5 column volumes with pure water. Equilibrate the column with a buffer system such as 1× PBS (pH 7.4) for 3-5 column volumes. Load the cell supernatant at a low flow rate for binding. After binding, wash the column with 1× PBS (pH 7.4) for 3-5 column volumes until UV absorbance returns to baseline. Elute the sample with 50mM acetate buffer (pH 3.5). Collect the elution peak under UV monitoring. Adjust the pH of the eluted product to 5-6 with 1M Tris-HCl (pH 8.0) and store it temporarily. Improve sample purity by replacing the buffer system with ultrafiltration, dialysis, or desalting columns, or by removing aggregates from the eluted product using molecular sieves. After purification, the protein that meets the purity requirements is sterile filtered using a 0.22 μm filter (Millipore, product number SLGVR13SL). After passing the SEC-HPLC purity test, it is packaged and stored at -80°C for future use.

[0896] 1.3.1 Preparation of Antibody Drug Conjugates

[0897] A 10-fold molar equivalent of TCEP (Tris(2-carboxyethyl)phosphine hydrochloride) was added to a 2-10 mg / ml antibody solution (1×PBS, 1 mM EDTA, pH 7.4), mixed and incubated at 37°C for 2 hours. A 15-fold molar equivalent of the drug-linker compound (prepared as described in Examples 22-45 or purchased) was then added, mixed and reacted at 25°C for 4 hours. After the reaction, the solution was ultrafiltered three times with 20 mM histidine pH 6.0 buffer to remove residual unreacted free small molecules. The sample purity was determined by SEC-HPLC, and the conjugation was determined by LC-MS.

[0898] 1.3.2 Method 2 for the preparation of antibody-drug conjugates (applicable to the preparation of ADC molecules with a DAR of approximately 4)

[0899] A 2.5-fold molar equivalent of TCEP (Tris(2-carboxyethyl)phosphine hydrochloride) was added to a 2-10 mg / ml antibody solution (1×PBS, 1 mM EDTA, pH 7.4), mixed and incubated at 37°C for 2 hours. An 8-fold molar equivalent of the drug-linker compound (prepared according to Examples 22-45 or purchased) was then added, mixed and reacted at 25°C for 4 hours. After the reaction, the solution was ultrafiltered three times with 20 mM histidine, pH 6.0 buffer to remove residual unreacted free small molecules. The sample purity was determined by SEC-HPLC, and the conjugation was determined by LC-MS.

[0900] 1.4 Antibody Drug Conjugate Purity Testing (SEC-HPLC)

[0901] The SEC-HPLC method was used to analyze the protein samples to characterize the molecular size uniformity of the recombinant protein and determine the purity of the recombinant protein. The HPLC used in this method was an Agilent 1260, the chromatographic column was a TSKgel G3000SWXL from Tosoh Bioscience, the mobile phase was 200mM phosphate buffer pH 7.0 containing 10% isopropanol, the detection temperature was 25°C, the flow rate was 0.5mL / min, the detection wavelength was 280nm, the target protein load was 50μg, and the analysis time was 40 minutes. For the SEC-HPLC data, the chromatogram was analyzed by manual integration, and the protein purity was calculated according to the area normalization method. The main peak was considered to be a monomer, the chromatographic peak before the main peak was called an aggregate, and the chromatographic peak after the main peak was called a fragment. The test results are shown in Table 4. Compared with the control ADC7, control ADC8 and control ADC9, the SEC purity of ADC-L26-4 (DAR8), ADC-L27-5 (DAR8), ADC-L22-3 (DAR8) and ADC-L23-3 was significantly improved.

[0902] Table 4 Purity analysis of antibody drug conjugates by SEC-HPLC

[0903] 1.5 DAR value detection of antibody drug conjugates (LC-MS mass spectrometry)

[0904] LC-MS mass spectrometry was used to analyze the ADC samples to characterize their DAR values. ~50 μg of ADC sample was added to 1 μl of PNGase F and incubated at 37°C for 4 hours. DTT was then added to a final concentration of 20 mM and incubated at 37°C for 1 hour. The sample was then centrifuged for analysis. The HPLC system used in this method was a Thermo Vanquish, the column was a Protein BEH SEC (Waters), the mobile phase was a 25% acetonitrile solution (containing 0.1% formic acid and 0.05% TFA), the flow rate was 0.2 mL / min, the detection wavelength was 280 nm, and the analysis time was 30 minutes. The high-resolution mass spectrometer used in this method was a Q Exactive Plus. The mass spectrometry parameters were as follows: Spray Voltage: 3.8 kV; Capillary Temp: 300°C; S-lens: 50; Sheath Gas: 35 arb; Aux Gas: 10 arb, Probe Heater Temp: 300°C; Full MS Scan range: 810-3500 m / z, Resolution: 17500; AGC: 3e 6 Maximum IT (maximum ion injection time): 200 ms; Microscans: 10; In-source CID: 20 eV. Mass spectrometry data were analyzed using Biopharma Finder 4.1, a mass spectrometry data analysis software. Deconvolution was performed using the universal Respect algorithm to calculate the molecular weights of the light and heavy chain mass spectrometry peaks and the mass spectrometry response signals of each component. The DAR value of the sample was calculated based on the corresponding mass spectrometry response values ​​of the sample-coupled small molecule.

[0905] After calculation, the test results of each ADC sample are shown in Table 5:

[0906] Table 5 DAR values ​​of antibody-drug conjugates detected by LC-MS mass spectrometry

[0907] ADC-L2-1 b This is an ADC sample prepared by coupling Trastuzumab with Example 25.

[0908] ADC-L6-1 a This is an ADC sample prepared by coupling Trastuzumab with Example 32.

[0909] ADC-L12-1 aThis is an ADC sample prepared by coupling Trastuzumab with Example 37.

[0910] 1.6 Hydrophobicity Testing of Antibody-Drug Conjugates (HIC-HPLC)

[0911] The HIC-HPLC method was used to analyze the test samples and characterize their DAR values. The HPLC used in this method was Agilent 1260, the chromatographic column was TSKgel Butyl-NPR from Tosoh Bioscience, the mobile phase A was 1.5M ammonium sulfate, 50mM phosphate buffer pH 7.0, the mobile phase B was 25mM phosphate buffer pH 7.0 containing 20% ​​isopropanol, the detection temperature was 25°C, the flow rate was 0.5mL / min, and the detection wavelength was 280nm. The test sample was first concentrated to 1mg / mL, and then 2M ammonium sulfate and 100mM phosphate buffer pH 7.0 solution were added to finally make the concentration of ammonium sulfate 0.8M, mixed evenly, and the supernatant was taken. The sample load was 40μg, and the mobile phase gradient was in 35 minutes. The mobile phase B increased from 0% to 70% within 30 minutes, and the analysis time was 50 minutes. For the HIC-HPLC data, the manual integration method was used to analyze the chromatogram, and the hydrophobicity was judged based on the peak time of the antibody. The test results are shown in Table 6:

[0912] Table 6 Hydrophobicity of antibody-drug conjugates detected by HIC-HPLC

[0913] Test Example 2 Plasma stability test of antibody conjugate

[0914] 2.1 Plasma stability test of antibody conjugates 1

[0915] A certain amount of ADC sample was added to sterile IgG-depleted human plasma to a final ADC concentration of 0.5 mg / ml. The samples were incubated at 37°C for 0, 3, and 7 days and then stored in a -80°C freezer. 50 μL of Protein A filler (MabSelect SuRe) was added to each tube and adsorbed using a shaker for 1 hour. After washing, elution, and Tris-HCl neutralization, the incubated ADC samples were obtained. LC-MS was used to analyze changes in DAR values ​​to determine the plasma stability of the samples. The test results are shown in Table 7.

[0916] Table 7 In vitro plasma stability of antibody-drug conjugates

[0917] The experimental results show that compared with the control ADC, the DAR value of the ADC disclosed in the present invention did not change significantly during the plasma incubation process, and had better stability in plasma.

[0918] 2.2 Plasma stability test of antibody drug conjugates

[0919] A certain amount of ADC sample was added to sterile IgG-depleted human plasma to a final ADC concentration of 0.5 mg / ml. The samples were incubated at 37°C for 0, 3, and 7 days and then stored in a -80°C freezer. 50 μL of Protein A filler (MabSelect SuRe) was added to each tube and allowed to adsorb using a shaker for 1 hour. After washing, elution, and Tris-HCl neutralization, the incubated ADC samples were obtained and their purity was determined by SEC-HPLC. The results are shown in Table 8.

[0920] Table 8 In vitro plasma stability of antibody drug conjugates

[0921] “-” means not detected.

[0922] The experimental results showed that compared with the control group, the ADC disclosed in the present invention had no significant change in SEC monomer purity during plasma incubation, and had better stability in plasma.

[0923] Test Example 3: Biological activity and related properties testing of antibody conjugates

[0924] 3.1 Anti-proliferative activity test of antibody-drug conjugates on tumor cells

[0925] 3.1.1. Anti-proliferative activity test of anti-Her2-ADC on tumor cells

[0926] Cells and materials: HER2-overexpressing human breast cancer cell line SKBR3 was purchased from ATCC, EMEM medium (ATCC#30-2003) was purchased from ATCC, McCoy's 5a medium (Gibco#16600-082), 1640 medium (Gibco#A10491-01), penicillin-streptomycin (Gibco#15140-122), and 0.25% Trypsin-EDTA (Gibco#25200-056) were purchased from Gibco (USA), human insulin (Solarbio#40112ES25) was purchased from Solarbio, 96-well plates (Greiner Bio-one#655098) were purchased from Corning (USA), and Cell-Titer Glo reagent (Promega#G7568) was purchased from Promega (USA).

[0927] Cell culture: SKBR3 cells were cultured in McCoy's 5a medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO2. Cells in the logarithmic growth phase were used for experiments.

[0928] Cell proliferation activity assay: The inhibitory activity of ADCs against SKBR3 cell proliferation was assessed using Cell-Titer Glo reagent. SKBR3 cells were dissociated from the cell culture flask and resuspended in fresh culture medium to adjust the cell density. 5000 SKBR3 cells were seeded in 96-well plates at a density of 90 μL / well and incubated overnight at 37°C, 5% CO2. The ADC was diluted to 5000 nM in complete culture medium and serially diluted 5-fold for a total of eight concentrations. Then, 10 μL of the diluted ADC solution was transferred to the 96-well plate, resulting in a starting and ending ADC concentration of 500 nM. The 96-well plate was incubated at 37°C, 5% CO2 for 3 days. Cell-Titer Glo reagent was then added to assess cell viability.

[0929] A negative control group (bottom) and a positive control group (top) were set up. The negative control group did not contain cells, but only the same volume of culture medium. All other procedures were the same as those for the experimental group. The positive control group did not contain the test antibody. All other procedures were the same as those for the experimental group.

[0930] Data Analysis:

[0931] Calculate the inhibition percentage (% Inhibition) and fit to obtain the IC of the compound 50 .

[0932] Percent inhibition (% Inhibition) = 1-100%*(Signal-Bottom) / (Top-Bottom).

[0933] Signal refers to the signal value of the experimental group, Bottom refers to the average signal value of the negative control group, and Top refers to the average signal value of the positive control group.

[0934] Experimental results:

[0935] Under the experimental conditions, the ADC disclosed herein exhibited strong proliferation inhibitory activity against the Her2-highly expressed human breast cancer cell line SKBR3.

[0936] Table 9 ADC anti-tumor cell proliferation activity (SKBR3 cell line)

[0937] ADC-L2-1 b This is an ADC sample prepared by coupling Trastuzumab with Example 25.

[0938] ADC-L6-1 a This is an ADC sample prepared by coupling Trastuzumab with Example 32.

[0939] 3.1.2. Anti-proliferative activity test of anti-Her2-ADC on tumor cells NCI-N87

[0940] Cells and materials: The HER2-overexpressing human gastric cancer cell line NCI-N87 was purchased from the Cell Bank of the Chinese Academy of Sciences, 1640 culture medium (Gibco#A10491-01), penicillin-streptomycin (Gibco#15140-122), and 0.25% Trypsin-EDTA (Gibco#25200-056) were purchased from Gibco (USA), 96-well plates (Greiner Bio-one#655098) were purchased from Corning (USA), and Cell-Titer Glo reagent (Promega#G7568) was purchased from Promega (USA).

[0941] Cell culture: NCI-N87 cells were cultured in 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. Cells were cultured at 37°C and 5% CO2. Cells in the logarithmic growth phase were used for experiments.

[0942] Cell proliferation activity assay: The inhibitory activity of ADC against the proliferation of the NCI-N87 cell line was assessed using the Cell-Titer Glo reagent. NCI-N87 cells were digested and dispersed from the cell culture flask, resuspended in the appropriate fresh culture medium, and the cell density was adjusted. NCI-N87 cells were plated at 6,000 cells / 90 μL / well in a 96-well plate and incubated overnight at 37°C, 5% CO2. The ADC was diluted to 5,000 nM in complete culture medium and a five-fold serial dilution was performed, with a total of eight concentration gradients. Then, 10 μL of the diluted ADC solution was transferred to the 96-well plate, resulting in an initial and final ADC concentration of 500 nM. The 96-well plate was incubated at 37°C, 5% CO2 for 3 days. Cell-Titer Glo reagent was added to assess cell viability.

[0943] A negative control group (bottom) and a positive control group (top) were set up. The negative control group did not contain cells, but only the same volume of culture medium. All other procedures were the same as those for the experimental group. The positive control group did not contain the test antibody. All other procedures were the same as those for the experimental group.

[0944] Data Analysis:

[0945] Calculate the inhibition percentage (% Inhibition) and fit to obtain the IC of the compound 50 .

[0946] Percent inhibition (% Inhibition) = 1-100%*(Signal-Bottom) / (Top-Bottom).

[0947] Signal refers to the signal value of the experimental group, Bottom refers to the average signal value of the negative control group, and Top refers to the average signal value of the positive control group.

[0948] Graphpad curve fitting was used to calculate the drug concentration at which the inhibition fraction was 50% as the absolute IC 50 .

[0949] Experimental results:

[0950] Under the experimental conditions, the ADC disclosed herein exhibited strong proliferation inhibitory activity against the human gastric cancer cell line NCI-N87 with high Her2 expression.

[0951] Table 10 ADC anti-tumor cell proliferation activity (NCI-N87 cell line)

[0952] ADC-L2-1 b This is an ADC sample prepared by coupling Trastuzumab with Example 25.

[0953] ADC-L12-1 a This is an ADC sample prepared by coupling Trastuzumab with Example 37.

[0954] 3.1.3 Anti-proliferative activity test of anti-Her2-ADC on tumor cells SKBR3

[0955] Cells and materials: The Her2-overexpressing human breast cancer cell line SKBR3 was purchased from ATCC, McCoy's 5a medium (Gibco#16600-082), penicillin-streptomycin (Gibco#15140-122), and 0.25% Trypsin-EDTA (Gibco#25200-056) were purchased from Gibco (USA), 96-well plates (Greiner Bio-one#655098) were purchased from Corning (USA), and Cell-Titer Glo reagent (Promega#G7568) was purchased from Promega (USA).

[0956] Cell culture: SKBR3 cells were cultured in McCoy's 5a medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO2. Cells in the logarithmic growth phase were used for experiments.

[0957] Cell proliferation activity assay: The inhibitory activity of ADCs against SKBR3 cell proliferation was assessed using Cell-Titer Glo reagent. SKBR3 cells were dissociated from the cell culture flask and resuspended in fresh culture medium to adjust the cell density. 5,000 SKBR3 cells were seeded in 96-well plates at a density of 90 μL / well and incubated overnight at 37°C, 5% CO2. The ADC was diluted to 100 nM in complete culture medium and serially diluted three-fold for a total of eight concentrations. Then, 10 μL of the diluted ADC solution was transferred to the 96-well plate, resulting in a starting and ending ADC concentration of 10 nM. The 96-well plate was incubated at 37°C, 5% CO2 for 3 days. Cell-Titer Glo reagent was added and cell viability was assessed.

[0958] A negative control group (bottom) and a positive control group (top) were set up. The negative control group did not contain cells, but only the same volume of culture medium. All other procedures were the same as those for the experimental group. The positive control group did not contain the test antibody. All other procedures were the same as those for the experimental group.

[0959] Data Analysis:

[0960] Calculate the inhibition percentage (% Inhibition) and fit to obtain the IC of the compound 50 .

[0961] Percent inhibition (% Inhibition) = 1-100%*(Signal-Bottom) / (Top-Bottom).

[0962] Signal refers to the signal value of the experimental group, Bottom refers to the average signal value of the negative control group, and Top refers to the average signal value of the positive control group.

[0963] Experimental results:

[0964] Under the experimental conditions, the ADC disclosed herein exhibited strong proliferation inhibitory activity against the Her2-highly expressed human breast cancer cell line SKBR3.

[0965] Table 11 ADC anti-tumor cell proliferation activity (SKBR3 cell line)

[0966] 3.1.4. Anti-proliferative activity test of anti-Her2-ADC on tumor cells NCI-N87

[0967] Cells and materials: The Her2-overexpressing human gastric cancer cell line NCI-N87 was purchased from the Cell Bank of the Chinese Academy of Sciences, 1640 culture medium (Gibco#A10491-01), penicillin-streptomycin (Gibco#15140-122), and 0.25% Trypsin-EDTA (Gibco#25200-056) were purchased from Gibco (USA), 96-well plates (Greiner Bio-one#655098) were purchased from Corning (USA), and Cell-Titer Glo reagent (Promega#G7568) was purchased from Promega (USA).

[0968] Cell culture: NCI-N87 cells were cultured in 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. Cells were cultured at 37°C and 5% CO2. Cells in the logarithmic growth phase were used for experiments.

[0969] Cell proliferation activity detection: Cell-Titer Glo reagent was used to detect the inhibitory activity of ADC on the proliferation of NCI-N87 cell line. NCI-N87 cells were digested and blown away from the cell culture flask, resuspended with the corresponding fresh culture medium, and the cell density was adjusted. NCI-N87 cells were 3000 cells / 90μL / well, seeded in a 96-well plate, and cultured overnight at 37°C and 5% CO2. The ADC concentration was diluted to 250nM with complete culture medium and a 4-fold gradient dilution was performed, with a total of 8 concentration gradients for each ADC. Then 10μL of the ADC dilution solution was transferred to the 96-well plate, that is, the initial and final concentration of the ADC was 25nM. The 96-well plate was cultured at 37°C and 5% CO2 for 5 days. Cell-Titer Glo reagent was added to detect cell activity.

[0970] A negative control group (bottom) and a positive control group (top) were set up. The negative control group did not contain cells, but only the same volume of culture medium. All other procedures were the same as those for the experimental group. The positive control group did not contain the test antibody. All other procedures were the same as those for the experimental group.

[0971] Data Analysis:

[0972] Calculate the inhibition percentage (% Inhibition) and fit to obtain the IC of the compound 50 .

[0973] Percent inhibition (% Inhibition) = 1-100%*(Signal-Bottom) / (Top-Bottom).

[0974] Signal refers to the signal value of the experimental group, Bottom refers to the average signal value of the negative control group, and Top refers to the average signal value of the positive control group.

[0975] Graphpad curve fitting was used to calculate the drug concentration at which the inhibition fraction was 50% as the absolute IC 50 .

[0976] Experimental results:

[0977] Under the experimental conditions, the ADC disclosed herein exhibited strong proliferation inhibitory activity against the human gastric cancer cell line NCI-N87 with high Her2 expression.

[0978] Table 12 ADC anti-tumor cell proliferation activity (NCI-N87 cell line)

[0979] 3.1.5 Anti-EGFR-cMet-ADC Anti-Proliferative Activity Test on Tumor Cells

[0980] Cells and Materials: HCT116 cells with intermediate EGFR expression and intermediate cMEt expression were purchased from ATCC under the catalog number CCL-247 and maintained in McCoy's 5a medium with 10% FBS. Capan-1 cells with low EGFR and low cMEt expression were purchased from Nanjing Kebai Biotechnology Co., Ltd. under the catalog number CBP60543 and maintained in IMDM medium with 20% FBS. PC9-hEGFR-Del19-T790M-C797S (PC9-DTC) cells with high EGFR and high cMEt expression were established by Crown Biotech (Taicang) Co., Ltd. and maintained in RPMI 1640 medium with 10% FBS and 0.5 μg / ml puromycin. HCC827 cells with high EGFR expression and intermediate cMet expression were purchased from ATCC under the catalog number CRL-2868 and maintained in RPMI 1640 medium with 10% FBS.

[0981] Cell proliferation activity assay: HCT116, Capan-1, PC9-DTC, and HCC827 cells were digested with trypsin (purchased from Gibco, Cat. No. 25200072), resuspended in their respective culture media, and the cell density was adjusted. The cells were seeded in 96-well plates (purchased from Corning, Cat. No. 3610) at the following cell densities: HCT116 cells 0.6×10 3 cells / 90μL, Capan-1 cells 4.0×10 3 cells / 90 μL, PC9-DTC cells 1.8×10 3 cells / 90 μL, HCC827 cells 3.0×10 3Cells / 90 μL were added and cultured overnight in a 37°C, 5% CO2 incubator. The next day, the antibody-drug conjugate was serially diluted with complete culture medium according to the experimental design, and 10 μL of the diluted antibody-drug conjugate was transferred to the corresponding wells of the plate. The cell plate was cultured in a 37°C, 5% CO2 incubator for 5 days. After 5 days, 50 μL of the antibody-drug conjugate was added to each well. Cell Viability Detection Kit (purchased from vkey-bio, product number A2010003N, for usage, refer to the product manual) was used to read the fluorescence value on an Envision instrument (purchased from PerkinElmer, model Envision 2105) to detect cell viability.

[0982] A positive control group and a negative control group were set up as 0% and 100% killing controls. The positive control group did not add the test drug, and other operations were consistent with the experimental group. The negative control group did not add cells, only added the same volume of culture medium, and other operations were consistent with the experimental group. The formula for calculating the cell proliferation inhibition rate is: inhibition rate = ((positive control-sample well reading value) / (positive control-negative well reading value)) × 100%. The inhibition rate of the corresponding drug in different cells was obtained by this formula, where the maximum inhibition rate is represented by Emax. GraphPad Prism 9.0 software was used to draw the graph and calculate IC 50 .

[0983] The results are shown in Table 13. The anti-EGFR-cMet-ADC drug has good proliferation inhibitory activity against tumor cells expressing EGFR and cMET in vitro.

[0984] Table 13 Inhibitory effects on tumor cell proliferation with different EGFR and cMET expression levels

[0985] 3.2 In vivo efficacy evaluation of anti-EGFR-cMet-ADC

[0986] 3.2.1. Efficacy Evaluation of Anti-EGFR-cMet-ADC in HCC827 Subcutaneous Tumor Model

[0987] Experimental reagents:

[0988] Lung cancer HCC827 cells: Shanghai Institute of Science, high expression of EGFR and cMet

[0989] RPMI-1640 culture medium: Gbico; Cat No.: A104910

[0990] Fetal bovine serum: Gibco; Cat No.: 10099-141C

[0991] 0.25% Trypsin-EDTA: Gibco, Cat No.: 25200-072

[0992] D-PBS (Calcium- and magnesium-free phosphate buffered saline): Hyclone, Cat. No.: SH30256.01

[0993] Matrigel:Corning,Cat.No.:356237

[0994] Experimental methods:

[0995] Animal information: Balb / c nude female mice, 5-6 weeks old, weighing approximately 14-20 g, were purchased from Beijing Weitonglihua Biotechnology Co., Ltd. The mice were housed in an SPF-grade environment with individual ventilation in each cage. All animals had free access to a standard certified commercial laboratory diet and free drinking water.

[0996] Cell Culture: Lung cancer HCC827 cells were cultured in RPMI-1640 (cell culture medium) supplemented with 10% fetal bovine serum and 1% Pen-Strep in a 37°C, 5% CO2 incubator. Twice weekly, cells were routinely digested and passaged using 0.25% trypsin-EDTA. When cell saturation reached 80%-90% and the required number of cells was reached, cells were harvested and counted.

[0997] Cell inoculation: 0.2 ml / (containing 1×10 7 HCC827 cell suspension (RPMI-1640:Matrigel, 1:1 volume ratio) was subcutaneously inoculated into the lumbar region of each mouse. Eight days after cell inoculation, mice were randomly divided into groups based on tumor volume for dosing, with grouping designated Day 0.

[0998] Dosing: ADC2-L19-1, control ADC10, and control ADC-ISO-2 were all administered at a dose of 1.5 mg / kg, via a single intravenous injection (IV) into the tail vein. Six mice were included in each group.

[0999] Tumor measurements and experimental parameters:

[1000] Tumor diameter was measured twice a week using a vernier caliper. Tumor volume was calculated using the formula: V = 0.5 axb 2 , a and b represent the long diameter and short diameter of the tumor, respectively. The body weight of mice was measured twice a week.

[1001] The anti-tumor efficacy of the compound was evaluated using the tumor growth inhibition rate (TGI) (%). TGI (%) = [(1 - (mean tumor volume of a treatment group at the end of dosing - mean tumor volume of the treatment group at the start of dosing) / (mean tumor volume of the solvent control group at the end of treatment - mean tumor volume of the solvent control group at the start of treatment)] x 100%.

[1002] Experimental results:

[1003] See Table 14, Figures 1 and 2.

[1004] Table 14. Tumor volume of HCC827 subcutaneous tumor model

[1005] Experimental conclusion:

[1006] In a mouse subcutaneous xenograft HCC827 tumor model, a single 1.5 mg / kg tail vein dose of the disclosed compound ADC2-L19-1 significantly inhibited tumor growth (P<0.0001). The compound was significantly superior to the control ADC10 and ADC-ISO-2 at equivalent doses (P<0.05, P<0.0001, respectively). The disclosed ADC molecule was not found to affect mouse body weight or cause any mortality at these doses, demonstrating good tolerance in mice.

[1007] 3.2.2. Efficacy Evaluation of Anti-EGFR-cMet-ADC in HCT116 Subcutaneous Tumor Model

[1008] Experimental reagents:

[1009] Colorectal cancer HCT116 cells: ATCC

[1010] McCOY'5A culture medium: Gbico; Cat No.: 16600-082

[1011] Fetal bovine serum: Gibco; Cat No.: 10099-141C

[1012] 0.25% Trypsin-EDTA: Gibco, Cat No.: 25200-072

[1013] D-PBS (Calcium- and magnesium-free phosphate buffered saline): Hyclone, Cat. No.: SH30256.01

[1014] Matrigel:Corning,Cat.No.:356237

[1015] Experimental methods:

[1016] Animal information: Balb / c nude female mice, 5-6 weeks old, weighing approximately 14-20 g, were purchased from Beijing Weitonglihua Biotechnology Co., Ltd. The mice were housed in an SPF-grade environment with individual ventilation in each cage. All animals had free access to a standard certified commercial laboratory diet and free drinking water.

[1017] Cell Culture: Intestinal cancer HCT116 cells were cultured in vitro using McCoy'5A (cell culture medium) supplemented with 10% fetal bovine serum and 1% Pen-Strep in a 37°C, 5% CO2 incubator. Twice weekly, cells were routinely digested and passaged using 0.25% trypsin-EDTA. When cell saturation reached 80%-90% and the required number of cells was reached, cells were harvested and counted.

[1018] Cell inoculation: 0.1 ml / (containing 2.5×10 6 HCT116 cell suspension (RPMI-1640:Matrigel, 1:1 volume ratio) was subcutaneously inoculated into the dorsum of each mouse. Seven days after cell inoculation, mice were randomly divided into groups based on tumor volume for dosing, with grouping designated Day 0.

[1019] Administration: The control ADC10 and ADC2-L19-1 were administered at a dose of 1.5 mg / kg via tail vein (IV) on day 0 and day 14. Six mice were included in each group.

[1020] Tumor measurements and experimental parameters:

[1021] Tumor diameter was measured twice a week using a vernier caliper. Tumor volume was calculated using the formula: V = 0.5 axb 2 , a and b represent the long diameter and short diameter of the tumor, respectively. The body weight of mice was measured twice a week.

[1022] The anti-tumor efficacy of the compound was evaluated using the tumor growth inhibition rate (TGI) (%). TGI (%) = [(1 - (mean tumor volume of a treatment group at the end of dosing - mean tumor volume of the treatment group at the start of dosing) / (mean tumor volume of the solvent control group at the end of treatment - mean tumor volume of the solvent control group at the start of treatment)] x 100%.

[1023] Experimental results:

[1024] See Table 15, Figures 3 and 4.

[1025] Table 15. Tumor volume of HCT116 subcutaneous tumor model

[1026] Experimental conclusion:

[1027] In a mouse subcutaneous xenograft HCT116 tumor model, both ADC10 and ADC2-L19-1 significantly inhibited tumor growth at 1.5 mg / kg administered twice (day 0 and day 14) through the tail vein (P < 0.01, P < 0.0001). ADC2-L19-1 was significantly superior to ADC10 (P < 0.05). The ADC molecules disclosed herein did not affect mouse body weigh...

Claims

1. A ligand-drug conjugate or a pharmaceutically acceptable salt thereof, having the general structural formula Pc-(LD) n , in, Pc is the ligand unit; D is the drug unit; L is a linker unit of the following structure, and the a end is connected to the ligand unit, in, X 1 , X 2 , X 3 Each independently selected from N or CR a1 , and X 1 , X 2 , X 3 At least one of N, the R a1 is selected from H, halogen, CN, C1-C3 alkyl, C2-C3 alkenyl, C2-C3 alkynyl, C3-C6 cycloalkyl or 4-7 membered heterocyclyl; n1 and n2 are each independently selected from 0, 1, and 2; X 4 , X 5 Each independently selected from C(═O), C(R b1 )(R b2 ), O, S, S(=O), S(=O)2, NR b3 or any combination thereof, the R b1 , R b2 , R b3 Each independently selected from H or R c1 , or R b1 , R b2 The carbon atoms to which they are attached together form a C3-C6 cycloalkyl or a 4-7 membered heterocyclic group, wherein the C3-C6 cycloalkyl or the 4-7 membered heterocyclic group is optionally substituted by one or more R c1 replace; Every R c1 Independently selected from halogen, CN, C1-C6 alkyl, C3-C6 cycloalkyl or hydrophilic group; L a Selected from R b4 , R b5 Each is independently selected from H, halogen, CN, C1-C3 alkyl, OH, O(C1-C3 alkyl), NH2, NH(C1-C3 alkyl) or N(C1-C3 alkyl)2, or R b4 , R b5 The carbon atoms to which they are connected together form a C3-C6 cycloalkyl group or a 4-7 membered heterocyclic group, m4 is selected from an integer of 1 to 10, L a The c-terminus of b connect; L b Select from chemical bonds or m5 is selected from an integer of 0 to 24, m6 is selected from an integer of 1 to 10, and each R b6 , R b7 , R b8 Each independently selected from H or R c6 , the R c6 is selected from halogen, CN, C1-C6 alkyl, C3-C6 cycloalkyl or hydrophilic group, L b The d end and L c connect; L c Selected from Y 1 -Y 2 -Y 3 , Y 1 Select from chemical bonds or m7 is an integer selected from 1 to 6, Y 2 is selected from a peptide residue consisting of 2-10 amino acids, Y 3 Selected from L c The b-end is connected to the drug unit D; n is a real number ranging from 1 to 16.

2. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein X 1 , X 2 All selected from N, X 3 Selected from CH.

3. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein n1 and n2 are each independently selected from 0 or 1; or, n1 is selected from 1 and n2 is selected from 0.

4. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein X 4 , X 5 Each independently selected from C(=O) or C(R b1 )(R b2 ), the R b1 , R b2 Each independently selected from H or R c1 , or R b1 , R b2 The carbon atoms to which they are attached together form a C3-C6 cycloalkyl or a 4-7 membered heterocyclic group, wherein the C3-C6 cycloalkyl or the 4-7 membered heterocyclic group is optionally substituted by one or more R c1 replace; or, X 4 is selected from C(═O), X 5 Selected from C(R b1 )(R b2 ), the R b1 , R b2 Each independently selected from H or R c1 , or R b1 , R b2 Together with the carbon atoms to which they are attached, they form a C3-C6 cycloalkyl or a 4-7 membered heterocyclic group, wherein the C3-C6 cycloalkyl or the 4-7 membered heterocyclic group is optionally substituted by one or more R c1 replace; or, X 4 is selected from C(=O), X 5 Selected from C(R b1 )(R b2 ), the R b1 , R b2 Each independently selected from H or R c1 , or R b1 , R b2 Together with the carbon atoms to which they are attached, they form a C4-C6 cycloalkyl group or a 4-6 membered heterocyclic group containing 1 O atom or 1 N atom, wherein the 4-6 membered heterocyclic group containing 1 O atom or 1 N atom is optionally replaced by one R c1 replace.

5. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, wherein L b Selected from chemical bonds, wherein m5 is selected from an integer of 1 to 16, or m5 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16, m6 is selected from an integer of 1 to 6, or m6 is selected from 1, 2, 3, 4, 5 or 6, or m6 is 2, R b7 , R b8 Each independently selected from H or R c6 , L b The d end and L c Connect; or, L b Select from chemical bonds or Where R b7 is H, and R b8 For R c6 , L b The d end and L c connect.

6. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, wherein each R c1 and R c6 Each is independently selected from halogen, CN, C1-C6 alkyl, C3-C6 cycloalkyl or a hydrophilic group, the hydrophilic group comprising one or more of the following atomic groups or structural fragments: C(=O), OH, O(C1-C3 alkyl), NH2, NH(C1-C3 alkyl), N(C1-C3 alkyl)2, C1-C4 alkyl-O, NHC(=O), N(CH3)C(=O), C(=O)NH2, S(=O)2NH2, C(=O)NH(C1-C3 alkyl), S(=O)2NH(C1-C3 alkyl), C(=O)N(C1-C3 alkyl)2, S(=O)2N(C1-C3 alkyl)2, or, Every R c1 and R c6 independently selected from halogen, CN, C1-C6 alkyl, C3-C6 cycloalkyl or W 1 -W 2 -W 3 -W 4 -W 5 , each W 1 , W 2 , W 3 , W 4 Each independently selected from a chemical bond, A-(C1-C4 alkyl-O) m1 or [A-(CR d1 R d2 ) m2 -NR d3 ] m3 -B, each A and B is independently selected from a chemical bond, C1-C 10 Alkylene, C3-C6 cycloalkylene, 4-7 membered heterocyclylene, O, S, NR d4 、C=O、C(=O)NR d5 、S(=O)2、S(=O)2NR d6 、S(=O)(=NR d7 ), or any combination thereof, the R d1 , R d2 , R d3 , R d4 , R d5 , R d6 , R d7 , R d8 Each is independently selected from H, C1-C6 alkyl, C3-C6 cycloalkyl, 4-7 membered heterocyclyl or (C1-C4 alkyl-O) k1 -C1-C6 alkyl, the C1-C6 alkyl, C3-C6 cycloalkyl or 4-7 membered heterocyclyl is optionally substituted by halogen, CN, OH, O(C1-C3 alkyl), NH2, NH(C1-C3 alkyl), N(C1-C3 alkyl)2, C(=O)NH2, NHC(=O)NH2 or COOH, m1 is selected from an integer of 0 to 30, m2 is selected from an integer of 1 to 5, m3 and k1 are each independently selected from an integer of 1 to 30, W 5 is selected from H, C1-C6 alkyl, C3-C6 cycloalkyl, 4-7 membered heterocyclyl, OH, O(C1-C3 alkyl), NH2, NH(C1-C3 alkyl), N(C1-C3 alkyl)2, C(=O)NH2, S(=O)2NH2, C(=O)NH(C1-C3 alkyl), S(=O)2NH(C1-C3 alkyl), C(=O)N(C1-C3 alkyl)2, S(=O)2N(C1-C3 alkyl)2, or, Every R c1 and R c6 Independently selected from W 1 -W 2 -W 3 -W 4 -W 5 , W 1 , W 2 are selected from chemical bonds, W 3 Selected from a chemical bond or A-(C1-C4 alkyl-O) m1 , W 4 Selected from A-(C1-C4 alkyl-O) m1 or [A-(CR d1 R d2 ) m2 -NR d3 ] m3 -B, each A and B is independently selected from a chemical bond, C1-C 10 Alkylene, C3-C6 cycloalkylene, 4-7 membered heterocyclylene, O, S, NR d4 、C=O、C(=O)NR d5 、S(=O)2、S(=O)2NR d6 、S(=O)(=NR d7 ), or any combination thereof, the R d1 , R d2 , R d3 , R d4 , R d5 , R d6 , R d7 , R d8 Each is independently selected from H, C1-C6 alkyl, C3-C6 cycloalkyl, 4-7 membered heterocyclyl or (C1-C4 alkyl-O) k1 -C1-C6 alkyl, the C1-C6 alkyl, C3-C6 cycloalkyl or 4-7 membered heterocyclyl is optionally substituted by halogen, CN, OH, O(C1-C3 alkyl), NH2, NH(C1-C3 alkyl), N(C1-C3 alkyl)2, C(=O)NH2, NHC(=O)NH2 or COOH, m1 is selected from an integer of 0 to 30, m2 is selected from an integer of 1 to 5, m3 and k1 are each independently selected from an integer of 1 to 30, W 5 is selected from H, C1-C6 alkyl, C3-C6 cycloalkyl, 4-7 membered heterocyclyl, OH, O(C1-C3 alkyl), NH2, NH(C1-C3 alkyl), N(C1-C3 alkyl)2, C(=O)NH2, S(=O)2NH2, C(=O)NH(C1-C3 alkyl), S(=O)2NH(C1-C3 alkyl), C(=O)N(C1-C3 alkyl)2, S(=O)2N(C1-C3 alkyl)2, or, Every R c1 Independently selected from A-(C1-C4 alkyl-O) m1 -W 5 or [C(=O)-CH2-NR d3 ] m3 -BW 5 , A is selected from a chemical bond or C=O, R d3 is selected from C1-C6 alkyl, B is selected from C1-C 10 Alkylene, C=O or a combination thereof C(=O)-C1-C 10 Alkylene, W 5 is selected from C1-C6 alkyl, C(=O)NH2 or S(=O)2NH2, m1 and m3 are independently selected from integers of 1 to 24; or, Every R c1 Independently selected from or, Every R c1 Independently selected from or, Every R c6 Independently selected from W 1 -W 2 -W 3 -W 4 -W 5 , W 1 , W 2 are selected from chemical bonds, W 3 Selected from a chemical bond or A-(C1-C4 alkyl-O) m1 , W 4 Selected from A-(C1-C4 alkyl-O) m1 or [C(=O)-CH2-NR d3 ] m3 -B, each A and B are independently selected from a chemical bond, a C1-C6 alkylene, NR d4 ,C(=O),C(=O)NH,S(=O)2,S(=O)2NR d6 、S(=O)(=NR d7 ), or any combination thereof, the R d1 , R d2 , R d3 , R d4 , R d6 , R d7 , R d8 Each is independently selected from H, C1-C6 alkyl or (C1-C4 alkyl-O) k1 -C1-C6 alkyl, m1 is selected from 0 to 24 An integer of , m2 is selected from an integer of 1 to 5, m3 and k1 are each independently selected from an integer of 1 to 24, W 5 Selected from C1-C6 alkyl, NH2, or, R c6 Selected from 7. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6, wherein L a Selected from R b4 , R b5 are each independently selected from H, m4 is selected from an integer of 1 to 6, L a The c-terminus of b Connect; or, L a Selected from R b4 , R b5 are each independently selected from H, m4 is selected from an integer of 1 to 6, L a The c-terminus of b connect.

8. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7, wherein L c Selected from Y 1 -Y 2 -Y 3 , Y 1 Select from chemical bonds or Y 2 A dipeptide, tripeptide or tetrapeptide residue selected from Val-Cit, Val-Ala, Gly-Gly-Phe-Gly, Ala-Ala-Ala, Y 3 Selected from L c The b-end of the drug unit D is connected; or, L c Selected from L c The b-end of the drug unit is connected to the drug unit D.

9. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein the linker unit L is selected from the following structures:

10. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 9, wherein the drug unit D is selected from a cytotoxic drug and a protein degrader.

11. The ligand-drug conjugate according to claim 10 or a pharmaceutically acceptable salt thereof, wherein the cytotoxic drug is selected from a tubulin inhibitor, a DNA damaging agent or a topoisomerase inhibitor, the tubulin inhibitor is, for example, a dolastatin, auristatin, maytansine, tubulysins or cryptomycins, the DNA damaging agent is, for example, a PBD, duocarmycin or calicheamicin, the topoisomerase inhibitor is, for example, a camptothecin; or, The cytotoxic drug is selected from camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotecan, rubitecan, actinomycin D, doxorubicin, duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin, etoposide, vincristine, vinblastine, paclitaxel, docetaxel, cabazitaxel, maytansine, maytansine-like, DM1, DM3, DM4, calicinomycin, MMAE, MMAF and Tubulysin D; or, The cytotoxic drug is selected from topoisomerase I inhibitors or auristatins; or The protein degrading agent is selected from GSPT1 protein degrading agents; or, The drug unit D is selected from or, The drug unit D is selected from 12. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein the general formula is Pc-(LD) n The ligand-drug conjugate or a pharmaceutically acceptable salt thereof is selected from the following compounds or a pharmaceutically acceptable salt thereof:

13. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 12, wherein the ligand unit Pc is selected from a polypeptide, an antibody or an antigen-binding fragment thereof; or, The ligand unit Pc specifically binds to one or more antigens selected from the following group: HER2 (ErbB2), p95HER2, HER3 (ErbB3), CD3, CD16, ROR1, DLL3, CDH6, CD70, CD5, CD20, BCMA, EGFR, VEGF, Trop-2, Claudin6, Claudin18.2, c-MET, CD30, MUC1, Nectin-4, CD22, CD74, CD19, CD79b, MSLN, TOPO2, EpCAM, CEACAM-5, Mesothelin, PD-L1, PSMA, ROR1, ROR2, CD25, CD33, CD123, FLT3, CD174, CD166, CD326, CD71, LIV- 1. MUC16, ENPP3, TDGF1, ETBR, TIM1, TIM3, LRRC15, CanAg / AFP, FAP, SLITRK6, KIT / CD117, STEAP1, SLAMF7 / CS1, NaPi2B / SLC34A2, GPNMB, AXL, B7-H3(CD276), PTK7 / CCK4, PRLR, E FNA4, 5T4, NOTCH3, CD142, CA6, GPR20, EphA2, LYPD3, FGFR2, FGFR3, FRα, CEACAMs, GCC, IntegrinAv, CAIX, P-cadherin, GD3, cadherin6, LAMP1, CD56, CD37, CD47, CD138 or CD352.

14. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 13, wherein n is selected from a real number of 1 to 8; or, n is selected from a real number of 2 to 8; or, n is selected from a real number of 6 to 8.

15. A linker unit represented by formula (L) for connecting a ligand unit and a drug unit to obtain a ligand-drug conjugate, in, X 1 , X 2 , X 3 , X 4 , X 5 , L a , L b , L c , n1 and n2 are as defined in any one of claims 1 to 8, the a-terminus of L is connected to the Ligand unit, and the b-terminus of L is connected to the Drug unit.

16. The linker unit according to claim 15, wherein the linker unit represented by formula (L) is selected from the following structural units:

17. A drug-linker compound represented by formula (II) or a pharmaceutically acceptable salt thereof, in, X 1 , X 2 , X 3 , X 4 , X 5 , L a , L b , L c , n1, n2 are as defined in any one of claims 1 to 8, and the drug unit D is as defined in any one of claims 10 to 11; X is selected from absent, O or NR e1 ; R 1 Selected from N(R e2 )(R e3 )、C1-C 10 Alkyl, C3-C 10 Cycloalkyl, 4-14 membered heterocyclic group, C6-C 10 Aryl or 5-10 membered heteroaryl; the C1-C 10 Alkyl, C3-C 10 Cycloalkyl, 4-14 membered heterocyclic group, C6-C 10 Aryl or 5-10 membered heteroaryl is optionally substituted with one or more R f Replace, each R f Independently selected from halogen, CN, =O, C1-C3 alkyl, OH, O(C1-C3 alkyl), NH2, NH(C1-C3 alkyl) or N(C1-C3 alkyl)2; R e1 , R e2 , R e3 Each is independently selected from H, C1-C6 alkyl, C3-C6 cycloalkyl or 4-7 membered heterocyclyl.

18. The drug-linker compound according to claim 17 or a pharmaceutically acceptable salt thereof, wherein X is selected from O; and / or R 1 is selected from C1-C6 alkyl or C3-C6 cycloalkyl, wherein the C1-C6 alkyl or C3-C6 cycloalkyl is optionally substituted by one or more R f Replace; or, R 1 is selected from methyl, tert-butyl or cyclopentyl.

19. The drug-linker compound or a pharmaceutically acceptable salt thereof according to claim 17, wherein the drug-linker compound or a pharmaceutically acceptable salt thereof represented by formula (II) is selected from the following compounds or pharmaceutically acceptable salts thereof:

20. A pharmaceutical composition comprising the ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 14 and a pharmaceutically acceptable excipient.

21. A method for treating mammalian tumors, comprising administering a therapeutically effective amount of the ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 14, or administering a therapeutically effective amount of the pharmaceutical composition according to claim 20 to a mammal, preferably a human, in need of such treatment.

22. Use of the ligand-drug conjugate according to any one of claims 1 to 14 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 20 in the preparation of a drug for treating tumors.

23. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 14 or the pharmaceutical composition according to claim 20 for treating tumors.

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